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In this study, the degradation system of <i>Shewanella oneidensis</i> MR-1 and goethite was constructed with chlorpyrifos as the target contaminant. The effects of initial pH, contaminant concentration, and temperature on the removal rate of chlorpyrifos during the degradation process were investigated. The experimental conditions were optimized by response surface methodology with a Box–Behnken design (BBD). The results show that the removal rate of chlorpyrifos is 75.71% at pH = 6.86, an initial concentration of 19.18 mg·L⁻¹, and a temperature of 30.71 °C. LC-MS/MS analyses showed that the degradation products were C₄H₁₁O₃PS, C₇H₇Cl₃NO₄P, C₉H₁₁Cl₂NO₃PS, C₇H₇Cl₃NO₃PS, C₉H₁₁Cl₃NO₄P, C₄H₁₁O₂PS, and C₅H₂Cl₃NO. Presumably, the degradation pathways involved are: enzymatic degradation, hydrolysis, dealkylation, desulfur hydrolysis, and dechlorination. The findings of this study demonstrate the efficacy of the goethite/<i>S. oneidensis</i> MR-1 complex system in the removal of chlorpyrifos from water. Consequently, this research contributes to the establishment of a theoretical framework for the microbial remediation of organophosphorus pesticides in aqueous environments.