A spectrofluorimetric method for the determination of chlorpyriphos in apples, based on the inhibition of the acetylcholinesterase enzyme (AChE), has been established. The method employs a non-fluorescent synthetic enzyme substrate (Indoxyl acetate), which is hydrolyzed by the enzyme to yield a highly fluorescent product. In presence of the inhibitor, the rate of formation of this fluorescent product is lowered The rate of change in the fluorescence is measured ΔF/Δt. (λex= 395 λem= 470 nm). The simple spectrofluorimetric method described for the determination of chlorpyriphos in apple has a limit of detection of 13.68 μM with relative standard deviation of 7.1%. The method was applied to chlorpyriphos added (28.5 and 57 μM) to apples with recoveries ranged between 107% and 91%.