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Background and aims: Synthetic organophosphates (OPs) inhibit acetylcholinesterase resulting in the accumulation of acetylcholine, failure of organs, and eventually death. Diisopropyl-fluorophosphatase (DFPase) is one of the OPs degrading enzymes that has broad substrate from OPs. In this study, for the first time, the secretory expression of DFPase in Bacillus subtilis was investigated in order to accelerate the biodegradation rate of OPs. Methods: DFPase gene was amplified using polymerase chain reaction (PCR) from the pET28-inaV/N-dfpase plasmid. The PCR product was subcloned in the pWB980 plasmid. Competent B. subtilis WB600 were transformed with recombinant plasmid. SDS PAGE technique was used to study the expression of protein secreted in superrich medium. Results: Appearance of the 946 bp band in agarose gel after digestion of transformed plasmid confirmed the presence of DFPase gene in this construct. Approximately, 35 kDa protein band was shown in culture medium after incubating at 35°C for 72 hours and 150 rpm. Measurement of enzyme’s activity was done by monitoring the release of fluoride from diisopropyl fluorophosphate (DFP), using ion-meter. Results showed that enzyme’s activity was 3333 U/L. Conclusion: Bacillus subtilis is a suitable host for production of secretory and active form of DFPase.