In this work, a method for the fast monitoring of OPs in flow-injection systems was evaluated. The fast Fourier transform continuous cyclic-voltammetry (FFTCCV) at the carbon-paste electrode in a flowing solution system was used for determination of OPs. In this method the S/N ratio is enhanced by using of fast Fourier transform of the analyte and signal integration. FFTCCV can be considered as a new sensitive, accurate and fast method for determination of drugs and some pesticides. However, in order to obtain better sensitivity for a specific target, experimental parameters should be optimized. Response surface methodology (RSM) was applied to optimize three effective parameters (enzyme activity, multiwall carbon nanotube quantity and acidic sol-gel quantity). The optimum values for the tested parameters were enzyme amount H0.169 U cm⁻², multiwall carbon nanotube (MWCNT) 0.607 mL and acidic sol-gel 1.012 mL. The optimum feed pH, feed flow rate, ATChCl concentration and sweeping-rate were found to be 7.4, 0.34 mL min⁻¹, 0.750 mM and 10Vs⁻¹, respectively. The long-term stability of this flow-through system was 80% of its initial response after 120 days. Based on an incubation time of 12 min, it was found that the detection limit for paraoxon was equal to 1.7×10⁻⁷ mg L⁻¹ (6.2×10⁻¹³ M). The developed biosensor exhibited good repeatability and reproducibility. This study provides a new, modern, sensitive tool for the analysis of organophosphate pesticides.