Time-resolved fluorescence spectroscopic measurements of second-generation new photosensitizer of hematoporphyrin monomethyl ether (HMME) were carried out in the physiological saline and different human serums by picosecond fluorescence lifetime spectrometer, respectively. The objective was to assess the fluorescence lifetime of HMME as a new enhanced contrast parameter between normal and malignant tissues for the photodynamic diagnosis of various cancers. The influence of emission wavelength and photosensitizer concentration, on the lifetime of HMME was also presented. No significant change in fluorescence decay was found between emission wavelength 625 and 690 nm. The lifetimes of HMME diluted in the physiological saline and human serum were about 14.6 and 16.6 ns, respectively. Experimental results also indicate a quite stable behavior with a decay time of 16.6 ns when HMME was diluted with different concentration. Time-resolved fluorescence spectrum of HMME can serve as a characteristic marker for optical biopsy of the malignant tissue because of the useful property of selective retention in malignant tumors after intravenous injection. Furthermore, the results in this study will contribute to the improvement of nanosecond fluorescence lifetime imaging method.