In this study, different approaches including addition of disulfide bridges, saturation of surface charges, and decrease of segmental flexibility have been used to increase thermostability of firefly luciferase. A disulfide bridge is introduced into Photinus pyralis firefly luciferase to make two separate mutant enzymes. One of the designed mutant (A103C/S121C) showed remarkable thermal stability, its specific activity decreased, whereas the A296C/A326C mutant showed tremendous thermal stability and 7.3-fold increase of specific activity. Analysis of enzyme according to residues B-factors shows that its C-terminal is much more flexible than its N-terminal. Two mutations in the most flexible region of luciferase were designed. Analysis shows that D476N mutation doesn' t have any significant effect but D474K mutation destabilized protein. On the other hand, flexibility analysis using dynamic quenching and limited proteolysis demonstrates that D474K mutation became more flexible than wild type. Protein engineering studies have shown that thermostable proteins have a higher frequency of Arg, especially in exposed states. To further elucidate the arginine saturation effects on stability of firefly luciferase, some of hydrophobic solvent-exposed residues in luciferase are changed to arginine. All of these residues are located at the external loops of L. turkestanicus luciferase.Introduction of double mutation (-Q35R/I232R) and triple mutation (- Q35R/I232R/I182R) were reserved specific activity of enzyme while its stability was enlarged and its flexibility was declined. Structural and functional properties of the mutants were investigated using different spectroscopic methods. It seems a clear relationship between stability and segmental flexibility in firefly luciferase controls its activity under different conditions.