Taking the plant-esterase from wheat bran as the enzyme source, comparing the physical adsorption method and the cross-linking with adsorption methods, and the glass fiber film as adsorption carrier, and then exploreed the optimum conditions for immobilization use enzyme activity recovery as the evaluation index. On the basis of single-factors experiments, the Box-behnken center-united experimental design principles were used to design the experiment and the response surface analysis of 3 factors (the buffer’s pH, immobilized time and immobilized temperature) and the interactive effect on immobilization. The optimum immobilized conditions were confirmed as followed: phosphate buffer’s pH 7.0, immobilized time 4 h, and immobilized temperature 29 ℃. Based on these conditions, the detection value of the enzyme recovery rate was 28.21%, and its verified value was 27.51%. The study provides a technical reference for rapid determination of organophosphorus and carbamate pesticide residues use immobilized wheat bran esterase.