Luciferase, bioluminescence enzyme, is one of the most useful enzymes for various applications in fieldsof biotechnology and molecular biology. Enhancing of thermostability of luciferase is required for theseapplications. Studies on hyperthermophile proteins have been shown that increasing thermal stability ofhyperthermophile proteins is related to their enhancing of the conformational rigidity. In the presentstudy, molecular dynamics simulation was carried out to assess the molecular stability and flexibilityprofile of the luciferase structure. Firefly luciferase is a protein with a large N-terminal and a small Cterminaldomain. Molecular dynamics simulation at two temperatures was used to investigate thecommon fluctuation sites in p.pyralis luciferase, which are considered to be thermally weak points. Twomutations (H489K and H489M) in the most flexible region of firefly luciferase were designed.Thermostability analysis showed that H489M mutation doesn t have any significant effect but H489Kslightly stabilizes the protein slightly. The optimum temperature for activity of H489K was increased.Intrinsic and ANS fluorescence studies also demonstrated that little structural changes for these twomutate proteins were occurred. Therefore, this study shows that replacing the histidine amino acid bylysine amino acid within flexible area will increase the thermalstability while the enzyme functionremains active.