In this paper a new method, that is, studying in vitro HpD two-photon fluorescence of cancer tissue using stimulation of femtosecond laser is reported. Femtosecond optical pulse generated by self-mode-locked Ti:sapphire laser stimulates cancer tissue of little mouse injected HpD. The spectrum of two-photon fluorescence of HpD is observed. But there is no such spectrum in the corresponding normal tissue. The experiment shows that the method will be an effective method in diagnosing cancer field.