She was educated in the Hawaiian Mission Academy, and taught Hawaiiana at Punahou School. Pukui was fluent in Hawaiian, and from the age of 15 collected and translated folk tales, proverbs and sayings. She worked at the Bishop Museum from 1938-1961 as an ethnological assistant and translator. She also taught Hawaiian to several scholars and served as informant for numerous anthropologists. She published more than 50 scholarly works. She is the co-author of the definitive Hawaiian-English Dictionary (1957, revised 1986), Place Names of Hawaii (1974), and The Echo of Our Song (1974), a translation of old chants and songs. Her book, Ōlelo Noeau, contains nearly 3,000 examples of Hawaiian proverbs and poetical sayings, translated and annotated. The two-volume set Nānā i ke Kumu, Look to the Source, is an invaluable resource on Hawaiian customs and traditions. She was a chanter and hula expert, and wrote lyrics and/or music to more than 150 Hawaiian songs.
In addition to her published works, Pukui's knowledge was also preserved in her notes, oral histories, hundreds of audiotape recordings from the 1950s and 1960s, and a few film clips, all collected in the Bishop Museum.
She was named a "Living Treasure of Hawaii" by the Honpa Hongwanji Mission of Hawaii in 1977.