Tyus, from Tennessee State University, participated in the 1964 Summer Olympics at age 19. In the heats of the event, she equalled Wilma Rudolph's World Record, propelling her to a favourite for the final, where her main rival would be fellow American Edith McGuire. Tyus won the final, beating McGuire by two tenths. At the same Olympics, she also won a silver medal with the 4 x 100 m relay team, finishing only behind Poland.
The following years, Tyus won numerous national championships in the sprint events, and a gold medal in the 200 m at the Pan-American Games. In 1968, she returned to the Olympics to defend her title in the 100 m. In the final, she set a new World Record to become the first woman to retain the Olympic 100 m title. Tyus also qualified for the 200 m final, in which she finished sixth. Running the final leg for the relay team, Tyus helped setting a new World Record, winning her third gold medal.
Tyus retired from sports after the 1968 Olympics.