Recently, biological effects induced by weak electromagnetic fields have been a public concern. Our previous study found temperature and electromagnetic field co-effects on insulin conformation. Therefore, in the present study, Raman spectroscopy was employed to investigate the secondary structure changes of insulin molecule induced by pulsed electric field (PEF) exposure at various temperatures. The content changes in alpha helix of insulin were obtained. Then, protein helix-random coil transition model was used to quantitatively study the experimental results. The theoretical model could figure out the effect of PEF on alpha helix contents of insulin at different temperatures. The protein secondary structure transits from helix to random coil evoked by PEF exposure and change of thermodynamic environment, which could explain the reason for the decline of alpha helix content of insulin caused by PEF exposure together with temperature rising. The results offer experimental basis and theoretical reference for further study of the mechanism of nonthermal effects of weak electromagnetic fields on biological molecule secondary structure.