In order to study the function and effect of staphylococcus aureus nitric oxide synthase, the full-length sequence of the gene was cloned, and the nitric oxide synthase overexpressing strain was constructed based on the nitric oxide synthase gene-deficient strain. Type validation was conducted to study the effect of NO on bacterial phenotype. The results showed that:the loss of nitric oxide synthase accelerated the rate of autolysis of bacteria, decreased the ability of anti-oxidation killing and the biofilm formation, both the loss and overexpression of nitric oxide synthase can affect the normal growth cycle and rate of bacteria. The different effects of exogenous NO on the biofilms of wild strains, knockout strains and overexpressing strains indicate that S. aureus nitric oxide synthase is also involved in the regulation of exogenous NO on biofilms. A series of phenotypic results indicate that the endogenous NO action of S. aureus not only blocks the Fenton reaction, enhances catalase activity, but also acts as a signaling molecule to regulate the transcription and expression of bacterial genes.