Secretion of recombinant enzymes to extracellular milieu is important for enhanced degradation of toxic pollutants since the substrates are often inadequately taken up by cells. The twin-arginine translocation (Tat) pathway is a secretion mechanism for the transport of folded proteins across the cytoplasmic membrane of bacteria. Notably, two substrate-specific Tat systems have previously been discovered in Bacillus subtilis. The uptake of organophosphates (OPs) is the rate-limiting factor in whole-cell degradation of OPs. In this study, to secret an OP-hydrolyzing enzyme, methyl parathion hydrolase (MPH), into the growth medium, the twin-arginine (RR-) signal peptide of trimethylamine N-oxide reductase (TorA) from Escherichia coli was used to target MPH to the Tat pathway of B. subtilis. Fractionation studies and MPH assays demonstrated that MPH was secreted into the culture supernatant where it was fully active. Export was fully blocked in a tat mutant, indicating that the observed export in wild-type cells was mediated exclusively by the Tat pathway. The amount of MPH present in the culture medium was estimated to be 6.1 mg/L. N-terminal sequencing of the purified MPH demonstrated that the TorA signal peptide had been processed correctly. The secretion of MPH neither inhibited cell growth nor affected cell viability. The recombinant strain showed the accelerated degradation for OPs and the culture supernatant effectively degraded OPs on vegetables. The recombinant strain may be ideal for large-scale production of MPH at low costs because of simplification of the protein purification step. The Tat pathway of B. subtilis was successfully utilized for extracellular secretion of MPH. This is the first demonstration of Tat-dependent export of an active heterologous protein in B. subtilis using an E. coli Tat signal peptide. This study highlights the potential of the B. subtilis Tat pathway for heterologous protein secretion.