The present work is focused on the application of an acetylthiocholine (ATCh) biosensor in a flow-injection system for the detection of organophosphorus pesticides. The optimal operating conditions of the flow-injection system were determined: flow-rate – 0.5mLmin −1 , substrate concentration – 100μM, incubation and reactivation time–10min. A calibration plot was obtained for ATCh concentration ranging from 20 to 200μM. A linear interval was detected along the calibration curve from 20 to 100μМ with a correlation coefficient R 2 =0.996. The sensitivity of the constructed biosensor was calculated to be 0.083μAμM −1 cm −2 .The application of the flow-injection system for detection and quantification of three organophosphorus pesticides – paraoxon ethyl, monocrotophos and dichlorvos in unary solutions and in binary mixtures was investigated as well. The inhibition curves for each pesticide was plotted and the linear intervals were determined along with the corresponding equations and detection limits – 0.87×10 −11 M for paraoxon, 1.08×10 −11 M for monocrotophos and 1.22×10 −10 M for dichlorvos. The bimolecular inhibition constants k i were calculated by performing amperometric measurements of the residual enzyme activity after incubation for 10min in a series of samples with varying pesticide concentrations (from 2 to 100μM). The highest inhibition potency was observed for paraoxon (2.3×10 5 M −1 min −1 ), and the lowest – for dichlorvos (3.5×10 4 M −1 min −1 ).The flow-injection system was used in the detection of anti-cholinesterase activity of two binary mixtures – paraoxon+monocrotophos and paraoxon+dichlorvos. It was interesting to observe that the total anti-cholinesterase activity of the mixtures was lower than the anti-cholinesterase activity of paraoxon with the same concentration in the sample.The storage stability of the enzyme membrane was considerably improved with respect to our previous work. After storage for 30 days, the enzyme membrane retained over 90% of its initial response. The half-life storage time of the enzyme membrane (50% residual activity) was almost tripled – from 25 to 75 days.