To investigate the relationship between the fluorescence spectra of serum and the brain injury effect, the alteration of fluorescence emission in serum was collected by fluorescence spectroscopy, the pathologic changes in the rat brain were obvious by histopathology after exposure to PEMF. Sprague-Dawley male rats were randomized into sham exposed and PEMFs exposed groups. After exposure to PEMF(3.5 ns rising time, 14 ns pulse width, and amplitude up to 200 kV/400 kV at 1 Hz repetitive rate) at 0.5, 1, 3, 6 and 12 h, the expression of S100B and the fluorescence spectra in serum were detected, the changes in brain morphology were observed. The results showed that the fluorescence intensity of 400 kV groups were higher than 200 kV groups at different time points after exposure to PEMF. It suggests that the extent of brain injury was associated with the pulse frequency. The trends of fluorescence spectra in serum coincide with the expression of S100B and pathologic changes. It shows that fluorescence spectroscopy could apply to analysis of the effect of brain injury after exposure to PEMF.