To investigate the diversity and dynamics of microorganisms in Chinese fresh beef (CFB) without acid discharge treatment during cold storage, high-throughput sequencing was employed to analyze the CFB refrigerated for 0, 3, 7, and 10 days. The results showed that the community richness of the fungi and bacteria decreased significantly. However, the diversity decreased in the early stage and increased in the later stage. At the phylum level, Ascomycota (74.1–94.1%) and Firmicutes (77.3–96.8%) were the absolutely dominant fungal and bacterial phyla. The relative abundance of both fungal and bacterial phyla displayed a trend of increasing and then decreasing. At the genus level, Candida (29.3–52.5%) and Lactococcus (19.8–59.3%) were, respectively, the dominant fungal and bacterial genera. The relative abundance of Candida showed a trend of increasing and then decreasing, while Lactococcus possessed the opposite trend. KEGG metabolic pathways analysis suggested that carbohydrate metabolism, membrane transport, and amino acid metabolism were the major metabolic pathways of bacteria. Bugbase prediction indicated the major microbial phenotype of bacteria in CFB during cold storage was Gram-positive (17.2–31.6%). Correlation analysis suggested that Lactococcus, Citrobacter, Proteus, and Rhodotorula might be the main microorganisms promoting the production of off-flavor substances in CFB. This study provides a theoretical basis for the preservation of Chinese fresh beef.