This study focuses on the identification of Peptoniphilus asaccharolyticus from spoiled fermented milk intended for students based on isolation, counting, morphological observation, biochemical identification, matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS), and whole-genome sequencing. The results showed that the isolate grew well on PCA medium under anaerobic conditions and was identified as P. asaccharolyticus on a fully automated microbial identification system with a confidence level of 98%. By MALDI-TOF MS, it was confirmed as P. sasaccharolyticus with a comparison value of 2.241. The result of whole genome sequencing showed an average nucleotide identity (ANI) of 98.58%. The proposed method allows rapid and accurate identification of P. sasaccharolyticus and can meet the requirements of food safety detection.