Abstract: Objective: Exploring the dynamic changes in the bacterial community during the industrial processing of fresh-cut lettuce. Methods: The bacterial composition of fresh-cut lettuce at different processing stages (raw material M, pre-washing PW, first washing W1, second washing W2, third washing W3, finished product P) was studied using high-throughput sequencing combined with traditional culture techniques. Results: The aerobic bacteria, coliforms and mould counts of lettuce decreased to 2.85±0.06 lg CFU/g, 1.01±0.03 lg CFU/g and 1.47±0.04 lg CFU/g after the four washes, which were lower than current standards of the enterprise Q/WWK0002S-2020. High-throughput sequencing results showed that at the phylum level, Proteobacteria was the dominant bacteria throughout the processing of fresh-cut lettuce, with a relative abundance of over 70%. At the genus level, the dominant bacteria in the of raw lettuce and pre-cleaning process were Acinetobacter, Pseudomonas and Alkanindiges. After the first NaClO cleaning, the genus Acidovorax became the dominant bacteria, all above 40%, followed by Pseudomonas and Acinetobacter. The differences in the microbial communities of the six samples could be seen in the relative abundance heatmaps and PCoA analysis. The microbial communities of samples M and PW were similar and grouped together. The microbial communities of samples W1, W2, W3 and P were more similar. The results of traditional culture showed that Raoultella ornithinolytica was the dominant bacteria throughout the processing of fresh-cut lettuce, followed by Acinetobacter spp. and Enterobacter bugandensis. Conclusion: A large number of microorganisms in the processing of fresh-cut lettuce came from the soil environment where the lettuce grew. Implementation of standardized planting to reduce microbial contamination at the source was the primary step to control the quality of fresh-cut lettuce, and at the same time, the strict control of cross-contamination during processing was also the key process to guarantee the quality. Defining the dominant spoilage bacteria such as Acinetobacter, Pseudomonas, and Acidovorax during processing was of great significance in formulating targeted and precise sterilization strategy to further enhance the safety of fresh-cut lettuce.