The present work studied an acclimation method for phosphorus accumulating organisms (PAOs) with a high content of acetone in culture solutions to develop microbial-based enzyme sensors for highly hydrophobic organophosphorus (OP) pesticides. Through three steps of cultivation and acclimation, only rod-shaped bacteria survived among the various PAOs. The extracellular enzymes released from the acclimated PAOs were salted out by using ammonium sulfate, then purified by a dialysis membrane and a DEAE-Sepharose FF anion exchange column. Two enzyme components were successfully separated—both of which showed hydrolase activity on disodium p-nitrophenyl phosphate (enzyme I, 1.57 μmol/(min·μg); enzyme II, 0.88 μmol/(min·μg) at 45°C). Further, SDS-PAGE gel electrophoresis results showed that the molecular weights of enzymes I and II were about 15.11 and 11.98 kDa, respectively. On this basis, the applicability of the enzyme in hydrophobic OP biosensors was demonstrated.