The laser-inducted fluorescence spectrum technology (LIF) was used for the first time to study the autofluorescence spectral characteristics of the heart, kidney, liver, fat, foreleg muscle, hind leg soleus muscle and musculus gastrocnemius of the rat performing motion exercises. The wavelength of the excitation light used during the measurement was in the range of 250-650 nm and the emission wavelength was 300-700 nm. When comparing the three-dimensional fluorescence spectra of the control group with those of the four groups of different motion states, a specific fluorescence peak related to the motion and located in the area where the excitation wavelength was (340±10) nm and the emission wavelength was (460±10) nm was found mainly in the spectra of the soleus muscle. From this fluorescence peak, it is possible to determine that its corresponding fluorescent substance is NADH (nicotinamide adenine dinucleotide reduced). When comparing the fluorescence spectra of the four groups of different motion modes, it was found that the motion mode has a conspicuous relativity with the peak intensity. The results show that the energy metabolism of the soleus muscle of the rat in motion is stronger than that of the foreleg, soleus muscle and other visceras, and the autofluorescence spectral characteristics of NADH form one of the effective indexes for determining the muscular metabolism degree.