Firefly luciferase (EC.1.13.12.7) from Photinus pyralis is a single polypeptide chain (62 kDa), responsible for emission of yellow-green (557 nm) light, known to be most efficient bioluminescence system that make it an excellent tool for reporter in nano-system biology. However, it is very sensitive to proteolytic degradation, which reduces its intracellular half-life, leads to loss in sensitivity and precision in analytic applications. In order to generate more stable luciferases against protease digestion, we substituted two tryptic sites: R(213), R(337) and also next residue to it (Q(338)) with another amino acids. Overall, all mutations brought about structural changes that indicated more compact structure upon mutation, which revealed by enhancement of tryptophan fluorescence, decreases flexibility and less surface hydrophobic pockets. In general, structural changes associated with a clear improvement in thermostability and resistance against trypsin hydrolysis. In particular, R337Q mutant shows higher light stability in mammalian cell culture, which makes it as a suitable reporter for imaging.