Objective: To provide a new idea for the development of Bacillus subtilis bio-preservative based on biofilm forming ability. Methods: The hydrophobic ability， self-agglutination rate， co-agglutination rate and biofilm forming ability of Bacillus subtilis were measured. The freshness and volatile flavor of tilapia fillets were measured by Bacillus subtilis. Results: The stronger the biofilm forming ability of Bacillus subtilis， the best preservation effect of tilapia fish. Bacillus subtilis can significantly reduce the content of volatile basic nitrogen in fish meat， so that the TVB-N content of fish was 24.43 mg/100 g on the third day of storage， which was lower than the control group (34.58 mg/100 g). Moreover，the production of alcohol， phenols， esters and acids， such as isoamyl alcohol， phenol， indole， acetic acid and other volatile flavor components in fish was significantly inhibited. At the same time， compared with the biofilm forming ability， the bacteriostatic ability of Bacillus subtilis was not correlated with the fresh-keeping ability. The results showed that the autoagglutination and biofilm forming ability of Bacillus subtilis were most correlated with the preservation ability of tilapia fillets， and could be used as a rapid index for screening Bacillus subtilis for preservation.