The esterase activity of Chrysemyswas determined by Ellman method, and the indole acetate was used as substrate to determine the esterase activity of Tupaiabelangeri. The optimum reaction conditions were determined by orthogonal array design.and the inhibition effects of several specific enzyme inhibitors were compared; The enzyme inhibition method was used to investigated the sensitivity of esterase form Chrysemyse and Tupaiabelangeri to organophosphorus and carbamate pesticides. The optimum conditions of esterase activity determination of Chrysemys were: incubation temperature 35 ℃, incubation time 15 min, buffer pH 7.5; The optimum conditions of esterase activity determination of Tupaiabelangeri were: incubation temperature 25 ℃, incubation time 15 min, buffer pH 8.0.The most sensitive to Chrysemysesterase was donepezil hydrochloride, the most sensitive to Tupaiabelangeri esterasewas bis (4-nitrophenyl) phosphate. The detection limits of esterase form Chrysemys and Tupaiabelangeri on trichlorfon, phoxim, dichlorvos, mevinphos, phosphamidon, metolcarb, carbofuran, methomyl, aldicarb and carbaryl were all lower than the maximum residue limits stipulated by national standards. The extracts of esterase from Chrysemys and Tupaiabelangeriwere showed high sensitivity to pesticide, which could be used as a new enzyme source in the enzyme inhibition method for rapid detection organophosphorus and carbamate pesticide.