This work provides new insights guiding the protein design of organophosphorus hydrolase. Through coupling kinetic measurements and molecular modeling, we found hotspot residues that enhance the stability of paraoxon in the binding site and produced OPH mutants exhibiting better paraoxon binding and catalytic properties. Two of the engineered mutants, D253E-H254R and D253E, enhanced the substrate binding and the catalytic activity of the enzyme significantly. Additionally, we have identified a stable binding mode of paraoxon that may help other researchers interested in modeling OPH-substrate interactions. We expect that our structural findings highlighting the importance of the residues surrounding the active site of OPH and their strong interactions will assist future research. Combined with work on other important and extensively studied active site residues, these results could help further improve the catalytic function of OPH and its variants.