Secondary structure of proteins is closely related to the overall quality of meat. The sequence of the changes in chemical bonds caused by secondary structure modification and rapid detection method of secondary structure fractions was investigated by near-infrared hyperspectral imaging combined with generalized (G2D-CS) and heterospectral (H2D-CS) two-dimensional correlation analysis in this study. Synchronous and asynchronous spectral results from G2D-CS analysis indicated that, as alpha-helix fraction decreasing, the spectral intensity of N-H stretching changed before that of the C-H and O-H, and the spectral fluctuation of O-H stretching occurred after the carbonyl related C-H band and before those aliphatic and aromatic C-H band. Feature wavebands identified by H2D-CS analysis obtained good results with R-CV(2) of 0.836 in predicting a-helix fractions. The results of this study are useful for interpreting the secondary structure modification process of proteins during frozen storage and monitoring the secondary structure fraction in a rapid way.