Lefkow was born in Nemaha County, Kansas. She attended Wheaton College as an undergraduate, and obtained her Juris Doctor (J.D.) degree at Northwestern University law school in 1971. After graduation, she became a law clerk for Thomas E. Fairchild. From 1982 to 1997 she was a United States magistrate, and from 1997 to 2000 a U.S. Bankruptcy Judge for the Northern District of Illinois.
In May 2000, Judge Lefkow presided over the enforcement of a high-profile trademark infringement case against the World Church of the Creator, an organization run by white supremacist leader Matthew F. Hale. The TE-TA-MA Truth Foundation, a peaceful, multicultural church in Oregon, sued Hale's church for using the name Church of the Creator. Some argued against the validity of the suit because Hale's church had been using the name since 1973, but the Foundation had trademarked it in 1987, and no contest was filed against the trademark within a five-year period, making their ownership legally incontestable. In January 2002, Lefkow ruled in favor of Hale, but her decision was overturned on appeal. On July 25, Lefkow ruled against Hale, saying that his church violated the copyright of the Church of the Creator by copying their name and infringing on their registered trademark. (Hale's organization has since been renamed the Creativity Movement)
An injunction was issued on November 19 forbidding Hale's church from using the term "Church of the Creator." Hale's World Church of the Creator was ordered to stop using the name on the Internet and to remove or cover up the phrase "Church of the Creator" on all of Hale's publications and products. In response to this decision, Hale sued Lefkow on December 24, falsely claiming that her order violated the Constitution in requiring the destruction of the group's bibles. Around this time, threats were made against Lefkow on the internet and her home address and family photographs of her husband and children were posted on the Stormfront.org website.
Meanwhile, an undercover FBI informant taped a conversation with Hale where he asked about Lefkow's home address and discussed her impending "extermination". On January 8, 2003, Hale was arrested on charges of plotting to murder Lefkow. The FBI informant in that trial received several death threats, and Lefkow initially was protected by a detail of the United States Marshals Service.
On April 24, Judge Lefkow ruled that the Creativity Movement had failed to stop using the name "World Church of the Creator" and should be fined $1,000 a day until it complied.
On April 6, 2005, Hale was sentenced to a 40-year prison term for soliciting an undercover FBI informant to kill Judge Lefkow.
On February 28, 2005, Lefkow returned home to find the bodies of both her husband and mother dead in the basement of her North Side home. According to an anonymous federal source, both Michael F. Lefkow, 64, and Donna Humphrey, 89, had been shot multiple times. The Cook County medical examiner's office stated that the victims were killed with .22 caliber shots to the head. No weapon was found at the scene, but two .22-caliber casings were recovered; evidence of a break-in was found as well. As of March 1, it was not known if Hale or his followers were involved in the murders, but police were investigating the possibility that hate groups were involved. On March 4, the FBI announced a $50,000 reward for information leading to the identification of anyone involved in the slayings. On March 8, investigators announced that DNA samples were obtained from a cigarette butt found inside the kitchen sink. Further evidence was recovered in and around the home, including a fingerprint, a bloody footprint, and a soda can.
Judge Lefkow and her children were again placed under the protection of the United States Marshals Service.
On May 18, 2005, Judge Lefkow testified before the U.S. Congress on the problem of providing security for judges, placing some of the blame for the attack on her family on rhetoric against judges issued by persons such as Pat Robertson.
On March 10 , 2005, Chicago Police and federal agents announced a possible break in the case. According to investigators, a van was stopped during a routine traffic stop in West Allis, Wisconsin, at 6 p.m. on March 9. As West Allis police officer Rick Orlowski approached the vehicle, the driver, identified as electrician Bart Ross, shot and killed himself. Later that night, a suicide note was found in the car which admitted to the murders of Lefkow's husband and mother, allegedly providing details about the crimes which would have been known only to the actual murderer. Ross was a plaintiff in a medical malpractice case that Lefkow had dismissed. Investigators also found over three hundred .22 caliber shells in the vehicle, casings of the same caliber that were found in the Lefkow home. DNA evidence from Ross allegedly matches the cigarette butt found in Lefkow's home. Ross also sent a handwritten letter to WMAQ-TV describing breaking into the Lefkow home with a plan to kill the judge.