In order to improve the production process of sour meat, a traditional fermented meat product of some ethnic minorities in China, this work studied the effect of commercial acidic protease on the microbial structure, physicochemical characteristics and flavor quality of sour meat during its fermentation. Using high-throughput sequencing, the bacterial and fungal community compositions were analyzed. The results showed that the addition of protease affected the relative abundance of Lactobacillus, Bacillus and Staphylococcus during the fermentation of sour meat, reduced fungal species richness, and promoted the release of free amino acids (FAAs). Gas chromatography-mass spectrometry (GC-MS) analysis showed that the addition of protease increased the number and contents of volatile compounds, such as alcohols, acids, esters, ketones and aldehydes in ripened sour meat. Correlation analysis showed that Staphylococcus was correlated positively with 3-hydroxy-2-butanone and Bacillus was correlated positively with 1-octen-3-ol. The sensory evaluation showed protease addition improved the taste, smell and overall acceptability of sour meat samples. Therefore, acidic protease has an important effect on the microbial community composition and flavor quality of sour meat, which provides a theoretical basis for improving the production process of sour meat.