This study used Fourier transform infrared (FTIR) spectroscopy and near-infrared (NIR) spectroscopy to analyze 66 beef samples heated for 0–15 min at 60 to 120 ℃, and revealed the dynamic evolution of protein structure by twodimensional correlation spectroscopy (2D-CoS). The results showed that as the heating temperature increased, the absorption peaks of amide A and amide I bands shifted, and the N–H and C–N vibrations increased. Moreover, the relative content of β-sheet increased and the relative content of β-turn decreased. The sequence of changes in the secondary structure of proteins was β-turn, β-sheet, α-helix and random coil. In addition, a predictive model for protein secondary structure (α-helix, β-sheet, β-turn and random coil) contents was constructed based on NIR spectroscopy data, which was demonstrated to have high predictive ability (corrected correlation coefficient > 0.9). In conclusion, this study provides an effective technical approach for fast structural analysis of proteins in meat products without the need for complex sample pretreatment.