Based on the principle of enzyme inhibition, a novel and sensitive lipase biosensor to determine organophosphorus pesticide is presented. Contact of the enzyme with pesticide samples results in specific inhibition of enzyme activity. Sensor calibration was possible by correlating the inhibition of enzyme activity with various concentrations of pesticide compound in a buffer solution. The sensor was successfully used to determine pesticide concentrations ranging from a low of 167ng/ml to 1.34μg/ml, and the detection limit is 81ng/ml. The effects of temperature, pH value, incubation time and solvent were also investigated. The sensor was also applied to the determination of dimethoate residues in the peel and flesh of tomato.