A novel time-resolved two-photon excitation fluorescence spectroscopic system based on a high repetition rate streak camera and capable of simultaneous measurement of fluorescence spectrum and lifetime of the samples is presented. The system has high temporal (6.5—200 ps) and spectral (1—3 nm) resolutions and permits rapid data acquisition and reliable and reproducible lifetime and spectrum determinations. The system is tested with standard fluorescent dyes (rhodamine 6G, coumarin 314 and their mixtures) and the lifetimes and spectra obtained agree well with those reported in the literatures. Experimental results indicate that our system provides a unique contrast mechanism for discriminating multi-fluorophore or multi-component of biological tissues through spectrally and temporally resolved information and may find extensive applications in multispectral fluorescence lifetime imaging and fluorescence resonance energy transfer imaging.