Two-photon fluorescence microscopy is a kind of nonlinear optical microscopy, which has the advantages of high time resolution, spatial resolution, high signal to noise ratio and intrinsic three-dimensional ability. Traditional two-photon fluorescence microscopy generally adopts tunable ultra-short pulse laser with pulsewidth of 100 fs as the light source. At present, two-photon fluorescence microscopy is further studied, and light source and detection method improvements are common means. In this paper, we introduce and summarize the recent advances in multicolor two-photon excited fluorescence microscopy and its application in biomedicine. Firstly, traditional application of femtosecond laser and optical parametric oscillator in multicolor imaging is introduced. Secondly, we focus on the application of fiber supercontinuum in multi-color microscopic imaging. Finally, we make a description of our recent work on continuum spectrum generation by enhanced self-phase modulation and selective excitation to achieve multicolor imaging. Multicolor two-photon imaging technology has the advantages of simple set-up, simple operation, and it has been broadly applied in biomedical and material science. Which provides a powerful tool and platformfor biomedical diagnosis and research.