Abstract: Objective: In this study, a colloidal gold immunochromatographic four lines card for the simultaneous determination of chlorpyrifos, fipronil, carbofuran and carbendazim in fruits and vegetables was prepared. Methods: The four lines card was prepared by using nitrocellulose membrane as carrier, pesticide competitive antigen as coating antigen and colloidal gold as labeling material. The parameter conditions, including concentrations of antibody for labeling, concentration of the coating antigen, sample extractant and extraction time were optimized. Under the optimal condition, the sensitivity, specificity and stability, as well as the accuracy of the four lines card were evaluated. Results: In the optimized of four lines card preparation process parameters, the concentration of colloidal gold labeled antibody of chlorpyrifos, fipronil, carbofuran and carbendazim were 3.2, 2.4, 3.2 and 3.2 µg/mL, and the concentration of antigen coating of T₁, T₂, T₃ and T₄ were 0.65, 0.80, 0.65 and 0.5 mg/mL, respectively. The limit of detection of chlorpyrifos was 0.1 mg/kg, fipronil was 0.02 mg/kg, carbofuran was 0.02 mg/kg, and carbendazim was 0.05 mg/kg. Except that the cross-reaction rates of carbofuran with carbosulfan was 20%, the cross-reaction rates of chlorpyrifos, fipronil and carbendazim with six structural analogues were less than 5%. It could be stored at room temperature for at least 12 months. Furthermore, 60 samples were analyzed and the detection results were consistent with those obtained in reference method. Conclusion: The four lines card prepared in this study was simple, accurate, fast and stable, which was suitable for the rapid screening and detection of chlorpyrifos, fipronil, carbofuran and carbendazim residues in fruits and vegetables.