Triazophos is a kind of organophosphorous pesticide which was widely used by farmers all over the world in 1990's. It is effective in controlling pesticide but harmful to human beings. Bioremediation is an effective and economic method to treat environment that has been polluted by hazardous organic compounds, so researchers paid much attention in this area. Most of which focused on isolating functional bacteria, studying its degrading mechanism, and cloning degradation-related genes. MP-4 was isolated from soil polluted by Triazophos for a long time and identified as Ochrobactrum sp.. The triazophos hydrolase (tpd) gene was cloned by the method of shotgun cloning, and the sequences were determined and analyzed. In the former tests it was found that there was only 18 base pairs different in tpd gene from mpd gene, which was isolated from methyl parathion degrading strain Pseudomonas putida DLL-1. Enzyme TPD can hydrolyze triazophos and methyl parathion while MPD cannot hydrolyze triazophos. Pseudomonas putida KT2440 is a metabolically versatile saprophytic soil bacterium that has been certified as a biosafety host for the cloning of foreign genes. This bacterium is known for its diverse metabolism and potential for development of biopesticides and plant growth promoters because of its ability to colonize rhizosphere of crop plants. Tpd gene was isolated from the genomic DNA of Ochrobactrum sp. MP-4 by PCR amplification. Recombinant plasmids pTPD was constructed by ligating tpd gene into broad host vector pBBRMCS-5. With the help of plasmid pRK2013, pTPD was transferred into Pseudomonas putida KT2440 to construct KT2440-DOP. KT2440-DOP can degrade many organophosphate pesticides and aromatics compounds. The specific activity of organophosphate hydrolase of KT2440-DOP was approximately 2 times of MP-4. Later, parameters affecting bioremediation of Organophosphate pesticide in soil using KT2440-DOP will be studied.