The invention relates to a femtosecond laser two-photon fluorescent biological microimaging system and an imaging method thereof, and aims at solving the problem that the cost is high and the imaging speed requirements cannot be met in an existing two-photon fluorescent microscope. A femtosecond laser device is easily unlocked due to the influence of external environment, and the unlocked laser device cannot excite a sample to generate a two-photon fluorescent signal; the two-photon fluorescent microimaging is imaging on the specific components of the sample and not complete imaging on the sample. The femtosecond laser two-photon fluorescent biological microimaging system is characterized by comprising a tunable femtosecond laser source Tsunami (1), a biological microscope (2), a spectrometer (3), a photomultiplier (4), a photodiode (5), a data acquisition card (6), an electrical translation platform (7), an electrical translation platform controller (8), a computer (9) and a beam splitting piece (10), wherein the biological microscope (2) comprises a reflection mirror M1 (11), a reflection mirror M2 (12), a dichroic mirror (13), an emission filter (14), an objective lens (15) and a condenser (16). The femtosecond laser two-photon fluorescent biological microimaging system is applied in the field of fluorescent microimaging.