Abstract: A novel and selective method based on inhibition reactions coupled with a prior separation step consisting of analytical pervaporation has been designed and used in order to develop a method for the determination of diclorvos. The method is based on the inhibitory effect of this organophosphorous pesticide (low boiling point 35°C) on the acetylcholinesterase catalysis after selective pervaporation of the analyte, The derivatization reaction is a two-step reaction involving choline oxidase (ChOD) and horseradish peroxidase (POD) with fluorimetric detection at lambda(ex) = 310 nm and lambda(em) = 415 nm of the dimer formed, The method applied to liquid samples has a linear determination range of 0.05-0.5 mmol L-1 (n = 8, r2 = 0.9959) with excellent precision (RSD<3.2%) and a sampling frequency of 3 h-1. When applied to solid samples the method has a linear determination range of 0.001-0.04 g kg-1 (n = 5, r2 = 0.9909, RSD<4.2%) and a sampling frequency of 2 h-1. The approach has been applied to the determination of diclorvos in natural water and spiked soil samples with recoveries between 89.9 and 109.7%.