This study aimed to investigate the effect of different thermal processing treatments on the protein digestion characteristics of beef. The beef samples were subjected to different cooking methods, namely steaming, boiling, and roasting, and different core temperatures (75 °C, 80 °C, 85 °C, and 90 °C), and were subjected to in vitro gastrointestinal digestion simulation. All the thermal processing treatments increased the protein digestibility; the samples that were steamed at 85 °C (S85), boiled at 80 °C (B80), and roasted at 80 °C (R80) showed the biggest gains. The S85 released more peptide species after gastrointestinal digestion, according to peptididomic studies. These differences were closely related to protein structure. Thermal processing treatments resulted in a higher degree of proteolysis and looser protein conformation, as evidenced by decreased intrinsic fluorescence and electrophoretic band intensity, increased surface hydrophobicity, and the change in protein secondary structure from α-helix to β-sheet and random coil. Based on the results, S85 was identified as the optimal thermal processing treatment for enhancing the digestibility of beef protein. The results provide valuable insights into the nutritional qualities and digestion of heat-processed beef protein.