Dengue hemorrhagic fever cases are high in Kota Pariaman West Sumatra and temephos is being used indiscriminately for its vector control. Present study is aimed to determine the susceptibility status and analyze the activity of acetylcholinesterase enzyme on Aedes aegypti larvae in Kota Pariaman West Sumatra. The susceptibility test was carried out using the WHO method, and biochemical tests of acetylcholinesterase (AChE) enzyme activity was done using the ELISA method. The mosquito larvae were found resistant to temephos using WHO diagnostic concentration (0.012 mg/L) and high resistant using ELISA method with absorbance values in living mosquitoes of 0.98–1.58 and dead mosquitoes of 0.92–1. The values of AChE enzyme activity in living mosquito larvae after insecticide exposure was 7.894 U/L, which is higher than dead larvae was 4.813 U/L and control larvae (without insecticide exposure) was 7.540 U/L.