Abstract: For the purpose of preparing anti-fenitrothion nanobodies with high-purity efficiently, the expression conditions of a recombinant anti-fenitrothion nanobody with low expression level in E. coli was optimized. The IPTG concentration and inducing temperature were selected as the independent variables and the nanobody expression level was used as the dependent variable for single-factor experiments, and the purification strategy was also investigated. The results showed that the highest expression level of 6 mg/L anti-fenitrothion nanobody was achieved at 37 ℃ without IPTG. Moreover, an interactive effect of IPTG dosage and inducing temperature was found. After a two-step purification of Ni affinity chromatography and gel filtration chromatography, the anti-fenitrothion nanobodies was finally yielded with more than 98% purity, indicating that the expression and purification strategies in this study can obtain anti-fenitrothion nanobodies efficiently. An ic-ELISA assay was then established based on the obtained nanobody, with an IC₅₀ of 5.81 ng/mL and an LOD of 0.25 ng/mL, and a detection range of 0.78~43.07 ng/mL. The assay was applied to determine fenitrothion in Chinese cabbage and lettuce samples. The recovery rate of addition was between 93.3%~111.7%, and the coefficient of variation (CV) was between 2.3%~18.2%. The proposed assay based on anti-fenitrothion nanobody has high sensitivity, which can meet the requirements for monitoring of fenitrothion under the national standard, thus can be used for rapid screening of fenitrothion. This study laid a foundation for the development of efficient and rapid immunoassays for fenitrothion.