It is believed that he was born in Tutwiler, Mississippi, in Tallahatchie County, although some sources say his birthplace was near Clarksdale, in Coahoma County. He was the youngest of the 11 children of william Hooker (born 1871, died after 1923), a sharecropper and Baptist preacher, and Minnie Ramsey (born c. 1880, date of death unknown). In the 1920 federal census, william and Minnie were recorded as being 48 and 39 years old, respectively, which implies that Minnie was born about 1880, not 1875. She was said to have been a "decade or so younger" than her husband (Boogie Man, p. 23), which gives additional credibility to this census record as evidence of Hooker's origins.

Hooker's date of birth is a subject of debate; the years 1912, 1915, 1917, 1920, and 1923 have been suggested. Most sources give 1917, though at times Hooker stated he was born in 1920. Information in the 1920 and 1930 censuses indicates that he was born in 1912. In 2017, a series of events took place to celebrate the purported centenary of his birth.

The Hooker children were home-schooled. They were permitted to Listen only to religious songs; the spirituals sung in church were their earliest exposure to music. In 1921, their parents separated. The next year, their mother married william Moore, a blues singer, who provided John Lee with an introduction to the guitar (and whom he would later credit for his distinctive playing style).

At the age of 14, Hooker ran away from home, reportedly never seeing his mother or stepfather again. In the mid-1930s, he lived in Memphis, Tennessee, where he performed on Beale Street, at the New Daisy Theatre and occasionally at house parties.

He worked in factories in various cities during World War II, eventually getting a job with the Ford Motor Company in Detroit in 1943. He frequented the blues clubs and bars on Hastings Street, the heart of the black entertainment district, on Detroit's east side. In a city noted for its Pianists, guitar players were scarce. Hooker's popularity grew quickly as he performed in Detroit clubs, and, seeking an instrument louder than his acoustic guitar, he bought his first electric guitar.

Hooker's recording career began in 1948, when Modern Records, based in Los Angeles, released a demo he had recorded for Bernie Besman in Detroit. The single, "Boogie Chillen'", became a hit and the best-selling race record of 1949. Despite being illiterate, Hooker was a prolific lyricist. In addition to adapting traditional blues lyrics, he composed original songs. In the 1950s, like many black Musicians, Hooker earned little from record sales, and so he often recorded variations of his songs for different studios for an up-front fee. To evade his recording contract, he used various pseudonyms, including John Lee Booker (for Chess Records and Chance Records in 1951–1952), Johnny Lee (for De Luxe Records in 1953–1954), John Lee, John Lee Cooker, Texas Slim, Delta John, Birmingham Sam and his Magic Guitar, Johnny Williams, and the Boogie Man.

Beginning in 1962, Hooker gained greater exposure when toured Europe in the annual American Folk Blues Festival. His "Dimples" became a successful single on the UK Singles Charts in 1964, eight years after its first US release. Hooker began to perform and record with rock Musicians. One of his earliest collaborations was with British blues rock band the Groundhogs. In 1970, he recorded the joint album Hooker 'n Heat, with the American blues and Boogie rock group Canned Heat, whose repertoire included adaptations of Hooker songs. It became the first of Hooker's albums to reach the Billboard charts, peaking at number 78 on the Billboard 200. Other collaboration albums soon followed, including Endless Boogie (1971) and Never Get Out of These Blues Alive (1972), which included Steve Miller, Elvin Bishop, Van Morrison, and others.

Among his many awards, Hooker was inducted into the Blues Hall of Fame in 1980, and the Rock and Roll Hall of Fame in 1991. He was awarded the Grammy Lifetime Achievement Award in 2000 and has a star on the Hollywood Walk of Fame.

Hooker died in his sleep on June 21, 2001, in Los Altos, California. He is interred at the Chapel of the Chimes in Oakland, California. He was survived by eight children, 19 grandchildren, and numerous great-grandchildren.