Suantang beef is a traditional delicious Chinese food cooked in Suantang (ST, a sour soup fermented by microorganisms). However, the impact of ST on beef quality is unclear, and the process of ST beef lacks unified technical standards. In the presented study, we found that the additional amount of salt, cooking time, meat thickness, and beef–ST ratio significantly affect the quality of ST beef. After optimization, it was found that when salt addition was 1%, cooking time was 3 min, meat thickness was 2 cm, and beef–ST ratio was 40%, the color determined by colorimeter, texture determined by texture analyzer, and sensory scores of beef cooked by ST were improved compared with boiled beef. ST decreased the pH value and cathepsin L activity of beef, increased the content of organic acid, and changed the protein composition of beef. ST made the beef have higher hardness, and have better chewiness and cohesion. At the same time, ST reduced the disagreeable odors of beef and improved beef flavor. In addition, 88 volatile compounds were detected in ST beef by HS-SPME/GC-MS. According to odor, threshold, and odor activity value (OAV), 24 critical aroma-active compounds were confirmed in ST beef. This study provides a basis for the potential industrialized production of ST beef.