Abstract: The bacterial phase changes and the effect of dominant spoilage bacteria on quality were studied with traditional identification method and high-throughput sequence technique using Chinese traditional food soybean curd sheets as materials. Sterile soybean curd sheets were inoculated with three dominant bacteria and their combination. The total viable counts, total volatile base nitrogen (TVB-N), pH, sensory score, and texture characteristics of samples during normal temperature storage were measured periodically for spoilage potential. The scanning electron microscopy, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and Fourier transform infrared spectroscopy (FTIR) were used to determine the effect of spoilage bacteria on the protein. The results indicated the bacterial phases of soybean curd sheets changed obviously during storage, and Acinetobacter, Staphylococcus as well as Kurthia became the dominant bacteria after 2 days of storage. The selected representative spoilage bacteria were re-inoculated to the samples, one protease-producing Staphylococcus sciuri and the mixed bacteria had the strongest spoilage potential, because the TVB-N and viscosity increased greatly, while the hardness, springiness, and chewiness decreased significantly (P<0.05). Scanning electron microscopy revealed that the surface protein structure of the samples was distorted and many holes appeared. SDS-PAGE result showed that the band of soybean protein 7S subunit faded or disappeared, however, the bands below 25 kDa were deepened obviously and a new band appeared. FTIR showed that the soybean protein β-sheet structure decreased, while the β-turn structure increased. In conclusion, Staphylococcus sciur is identified as specific spoilage bacteria of soybean curd sheets during normal temperature storage, it provides a valuable reference for the spoilage bacteria control and prolong the product shelf-life.