In this study, the optimal reaction system of acetylcholinesterase ( AChE) in 6th instar larval Dendrolimus superans was determined by orthogonal matrix method, then sensitivities of AChE to DDVP, lambda-cyhalothrin and avermectins were compared through enzyme assay. The results showed that the optimal conditions of AChE activity assay in 6th instar larval D. superans were 0.2 g/ mL of AChE concentration, 0.8 mmol/ L of substrate concentration, pH 7.5, 35 c of temperature and 5 minutes of reaction time. Moreover, the three pesticides, DDVP, lambda-cyhalothrin and avermectins, showed different inhibition to in vitro AChE activity in 6th instar larval D. superans. Relationship of inhibitory effect and pesticides was in a concentration way. Among three pesticides, DDVP showed the most significant inhibition to AChE activity in 6th instar larval D. superans (IC50 = 3.018 ∙10 -4 mg/ mL), following lambda-cyhalothrin(IC50 =1.436 ∙10 -2 mg/ mL), whereas avermectin was the weakest. These results will provide basal information for further studying the biochemical characteristics of AChE and toxicity mechanisms of pesticides.