Objective To understand the influence of cultivation conditions on the formation of Salmonella biofilm and provide basis for ensure food safety.Methods Congo red staining and crystal violet staining method were used to screen biofilm （BF） formation strains from 400 foodborne Salmonella. Scanning electron microscope was applied to observe BF formation. Whole-genome sequencing analysis was used to identify BF formation related genes. Tube culture and microplate quantitative detection methods were used to determine BF formation of Salmonella under different culture conditions. The effect of different culture time， medium type， glucose content， peptone content and Vitamin B₁ （VB₁） content to Salmonella BF formation were studied.Results Eight （2.00%） BF-forming Salmonella isolates were obtained. Compared to LB medium， BHI was more suitable for BF formation for Salmonella. When cultured for 96 h， the BF formation of Salmonella on BHI was much more than that on LB. Fifteen BF-related genes including trx2， fadI， bsmA， bssS， tabA， bcsE， csgD， csgA， B， C， seqA， dam， fliZ， flhD and motB were detected in BJ44D， ZZSR2-2008， NYSR94-08 and yl19Ta. The best BF formation nutrient conditions for Salmonella were that 0.6% or 0.8% glucose， 4.0% peptone， and 50 mg/L VB₁ were respectively supplemented in BHI broth （P<0.05）.Conclusion It is indicated that different culture conditions significantly correlated with BF formation of Salmonella and have strain-specificity， and can provide theoretical basis for BF formation control of Salmonella in the food production chain.