Shiga toxin-producing Escherichia coli (STEC) is a zoonosis and food-borne pathogenic bacterium. It is necessary to understand its characteristics and potential pathogenicity and analyze the production ability of Shiga toxin (Stx) in the biofilm state. The traditional PCR was performed to amplify the stx variant gene subtype. The results showed that strain 1 carried stx1c+stx2d subtype, strain 2 carried stx1+stx2f subtype, and strain 3 carried stx2e subtype. The results of multilocus sequence typing (MLST) showed that the three strains were relatively distantly related and possessed genetic diversity. The crystal violet staining test and the cytotoxicity test of Vero cell were used to explore the biofilm-forming ability and Stx-producing ability at 4 ℃, 10 ℃, 25 ℃, and 37 ℃. It showed that temperature significantly affected the biofilm-forming ability and Stx-producing ability. All STEC strains had the strong ability of biofilm formation and Stx production at 37 ℃. The results of the cytotoxicity test of Vero cell showed that strain 2 had the weakest Stx-producing ability under the four different temperature conditions and strain 3 showed strong biofilm-forming ability and Stx-producing ability. Especially at 4 ℃and 10 ℃, strain 3 had the strongest biofilm-forming ability compared to strain 1 and stain 2. Retail meat in China is contaminated with STEC which could be potentially pathogenic. Especially with the discovery of strains with strong biofilm-forming ability and Stx-producing ability, we should strengthen the monitoring of STEC during food processing.