Abstract: To investigate the effects of cold plasma treatment on the oxidation and structure changes of myofibrillar proteins tilapia, the fish was treated by cold plasma with different time (0, 1, 2, 3, 4, 5 min) and different voltage (40, 50, 60, 70, 80 kV), respectively, the carbonyl content, sulfhydryl content, hydrophobicity, Zeta potential, SDS-PAGE, UV spectra, fluorescence spectra and Raman spectrometer of myofibrillar protein were measured after treatment. The results showed that the carbonyl content and surface hydrophobicity of myofibrillar protein were increased gradually, as the treatment time extending or voltage increased, while the total sulfhydryl and the active sulfhydryl content were decreased. When the treatment time was extending to 5 min, the total sulfhydryl and the active sulfhydryl content were decreased to 66.91 and 52.76 μmol/g, respectively. The carbonyl content increased from 1.23 to 2.16 nmol/mg, if the treatment voltage was increased to 80 kV. Cold plasma treatment decreased the absolute value of Zeta potential of myofibrillar protein. The myosin heavy chain band of myofibrillar protein was increased based on SDS-PAGE results. As treatment time extending and voltage increasing, the UV spectrum of myofibrillar protein was blue-shifted with an increased UV absorption intensity, and the fluorescence absorption intensity of protein was weakened. Based on the Raman spectrometer determination, the content of α-helix was increased, the contents of β-sheet and β-turn were reduced, and the random coils had no obvious changes in myofibrillar protein of tilapia after cold plasma treatment. In conclusion, extending the treatment time or increasing treatment voltage of cold plasma can accelerate the oxidation of myofibrillar protein to result in the changes of their second-order structure and conformation.