Abstract: 【Objective】The study aimed to elucidate the role and mechanism of double oxidase 1 gene（duox1）in the innate immune response of Procambarus clarkii against Staphylococcus aureus infection，providing technical support for the sustainable and healthy development of P. clarkii industry.【Method】The real-time fluorescence quantitative PCR was used to detect the relative expression of duox 1 gene in hemocytes，hepatopancreas，intestine and gill tissues of P. clarkii after stimulation of S. aureus. By using RNA interference（RNAi）to down-regulate the expression of the duox1 gene and subsequently stimulating with S. aureus，the survival rate of the P. clarkii was determined. The contents of H₂O₂ in the hepatopancreas were measured with an H₂O₂ detection kit. Melanization in the hemolymph was observed under an electron microscope. The expression of antimicrobial peptide genes（toll1、dorsal、crustin3和crustin4）in the hepatopancreas was assessed using real-time fluorescence quantitative PCR.【Result】After stimulation by S. aureus，the relative expression of the duox1 gene in hemocytes，hepatopancreas，intestine and gill tissues of P. clarkii showed an overall upward trend compared to the PBS group. This suggested that the duox1 gene may be involved in the innate immune response of P. clarkii against bacterial infection，with a potential role in combating bacterial invasion. Compared to the dsGFP+S. aureus group，after RNA interference and S. aureus stimulation，the survival rate of P. clarkii greatly decreased. The H₂O₂ content in the hepatopancreas showed a trend of initial reduction followed by recovery（reaching its lowest point 24 h after stimulation），and the degree of hemolymph melanization was markedly weakened. After duox1 gene expression was interfered and S. aureus infection，the expression of antimicrobial peptide genes（toll1、dorsal、crustin3和crustin4）in the Toll signaling pathway of the hepatopancreas was suppressed，leading to a reduction in innate immune capacity against bacterial infection，ultimately resulting in a decreased survival rate of P. clarkii.【Conclusion】The duox1 gene of P. clarkii participates in the innate immune response of the body by regulating H₂O₂ production，affecting the melanization of hemolymph，and regulating the expression of antimicrobial peptide genes such as toll1、dorsal、crustin3和crustin4，thereby assisting the body in resisting infection by S. aureus.