Growth and reproduction of specific spoilage bacteria contribute significantly to the spoilage of fresh-cut fruits and vegetables. In this paper, the change regulation of total colony count of fresh-cut lotus root during cold storage period was firstly analyzed, then the spoilage bacteria were separated by dilution-plate method, and the dominant spoilage bacteria were identified using 16S ribosomal DNA based bacterial identification system. The results showed that when the fresh-cut lotus root was preserved at 4 ℃ for 8 days, and its total colony count increased to 4.17×10⁵ CFU/g, which the early microbial spoilage was thought to be developed. Based on the homology analysis of 16S rDNA sequences, four dominant spoilage bacteria were eventually identified as Acinetobacter calcoaceticus, Klebsiella sp., Raoultella ornithinolytica and Enterobacter cloacae.