In this paper, three kinds of molecularly imprinted sol-gel film (MISF) which have good thermal and chemical stability and three-dimensional network porous structure have been developed. The material was synthesized by sol-gel technology using lambda-cyhalothrin, trichlorphon and beta-cyfluthrin as model analytes. The special structural behavior makes the material have good capability to seizure and capture template molecules which enhanced the MISF’s recognition efficiency and the property of separation and enrichment. The material was environmental friendly and easy to operate. The binding characteristic of the imprinted film to the three pesticides was evaluated by equilibrium binding experiments. The SEM of the three MISF has been performed to characterize the morphology of MISF. The MISFs were combined with chemiluminescence (CL) technology by modifying the sol-gel into 96 micro-well plate to establish one molecular imprinting- chemiluminescence (MI-CL) sensor which realized high selectivity, sensitivity and high throughput simultaneous assay of three pesticides. The proposed method was successfully applied to the simultaneous determination of multi-component pesticide residue in foodstuff.