The effect of pulsed electric field on molecular structure of soy protein isolate (SPI) was investigated by Raman spectroscopy method. The applied pulsed electric field was up to 50 kV·cm-1 with pulse width 40 μs. It was demonstrated from the Raman spectra that the PEF treatment under 50 kV·cm-1 had induced disappearance significantly of peak near 2 886 cm-1 bond. It was also explored that with the increase in treatment time, the polarity of microenvironment of aliphatic amino acid residues and the exposure of tryptophan residues from a buried hydrophobic microenvironment were increased. On the other hand, the interaction of serine acid residues, the C—H plane bend vibration, C—N stretch vibration, and the CO stretch vibration of aspartic acid and glutamic acid were decreased. The embeding or participation of the tyrosine phenolic groups as hydrogen bond donors was firstly increased with the treatment time (less than 1 600 μs), and afterwards decreased (from 1 600 to 3 200 μs).