The uptake of organophosphates (OPs) is a rate-limiting factor in whole-cell biocatalysis systems. Here, we report the periplasmic secretion of methyl parathion hydrolase (MPH) by employing the twin-arginine translocation (Tat) pathway in Escherichia coli. The twin-arginine signal peptide of trimethylamine N-oxide reductase (TorA) from E. coli was used for exporting MPH to the periplasm of E. coli, alleviating the substrate uptake limitation. A periplasmic expression vector, pUTM18, coding for TorA-MPH was constructed, and the periplasmic secretion and functionality of MPH were demonstrated by cell fractionation, immunoblotting, and enzyme activity assays. The strain expressing periplasmic MPH showed 3-fold higher whole-cell activity than the control strain expressing cytoplasmic MPH. Suspended cultures also exhibited good stability, retaining almost 100% activity over a period of 2 weeks. Owing to their high activity and superior stability, these "live biocatalysts" are ideal for large-scale detoxification of OPs.