Rutherfurd was born in Morrisania, New York to Robert Walter Rutherford and Sabina Morris, and was the grandson of U.S. senator John Rutherfurd. He graduated from Williams College, Massachusetts in 1834, then practiced law beginning in 1837 with William H. Steward in Auburn, New York. He was admitted to the bar in 1837. On July 22, 1841 he married Margaret Chanler. Their son Stuyvesant Rutherford was born in 1842.
Lewis Rutherfurd abandoned his study of law in 1849 to dedicate his leisure to science, particularly astronomy. He performed pioneering work in spectral analysis, and experimented with celestial photography. He invented instruments for his studies, including the micrometer for measuring photographs, a machine for producing improved ruled diffraction gratings, and the first telescope designed specifically for astrophotography.
Using his instrumentation, Rutherfurd produced a quality collection of photographs of the Sun, Moon, and planets, as well as star clusters and stars down to the fifth magnitude. In 1862 he began making spectroscopic studies using his new diffraction grating. He noticed distinct categories of spectral classes of stars, which Angelo Secchi expanded upon in 1867 to list a set of four stellar classes.
Rutherfurd served as a trustee of the Columbia University from 1858 until 1884, and donated his photographs to that institution. In 1884 he was named as one of the delegates to the International Meridian Conference. He was one of the original members of the National Academy of Sciences created in 1863, and was an associate of the Royal Astronomical Society.
In 1887 his health began to fail.
Richard Proctor, the greatest popularizer of astronomy in the nineteenth century, called Rutherford "the greatest lunar photographer of the age."