For the problem of weak exciting ability and short source life of xenon lamp in fluorescence imaging system, we proposed a new fluorescence excitation method that uses supercontinuum laser (SCL) to be the excitation source in bioluminescence imaging technology. The fluorescence imaging experiments of the fluorescence imaging test card by the two excitation sources were carried out and then the experiments at different SCL excitation power spectrum density were also completed. By the method of linear fitting and extrapolation, we analyzed the fluorescence excitation ability of the two sources at the same exposure time and power spectrum density. We obtain a conclusion that the fluorescence excitation intensity of SCL source is nearly twice of that of xenon lamp. Finally, through the analysis of the average and maximum photon density of the test perssad under two sources, the experiment results are proved again. The work is conducive to solve the problem of the source life and the fluorescence excitation ability of the light source in fluorescence imaging system.