In order to explore the effect of pH on the structure and thermal stability of myoglobin, the molecular structure and surface hydrophobicity of myoglobin at normal pH (5.6) and high pH (6.4) under refrigerated (4 ℃) and heating (72 ℃) conditions as well as its thermal denaturation degree were characterized by various spectroscopic techniques. The results showed that pH had no significant effect on the conformation of myoglobin at 4 ℃. However, the endogenous fluorescence intensity, protein surface hydrophobicity and thermal denaturation of myoglobin at 72 ℃ were significantly higher at pH 5.6 than at pH 6.4. The tertiary structure of myoglobin was more severely destroyed at pH 5.6 than pH 6.4, resulting in reduced thermal stability. Therefore, environmental pH can affect the thermal stability of myoglobin by altering its structural stability. High pH can increase the thermal stability of myoglobin, which is an important cause for the persistent pink color of dark, firm, dry (DFD) beef during cooking.