Nattokinase from traditional food natto has the potential to be a thrombolytic agent. Its terminal sequence has an important effect on the catalytic performance of nattokinase, but the specific mechanism is still unclear. In this study, computational simulation combined with truncated mutagenesis and alanine scanning technology were used to identify the key sites affecting the catalytic performance of nattokinase. Subsequently, mutants Q10L and Q275G with 4.00-fold and 4.83-fold increased half-lives at 55 °C were screened by site-directed saturation mutagenesis. Constraint network analysis and molecular dynamics simulation revealed that the thermal stability of the two mutants was enhanced by solvent interaction and indirect effects on the Ca