Luciferase is a general term for enzymes that produce biofluorescence in nature. Luciferase can catalyze the oxidation of luciferin to oxyluciferin. In the process of luciferin oxidation, biofluorescence is emitted. The biofluorescence released during the oxidation of luciferin can then be measured by a fluorometer. In our work, the luciferase came from the firefly Photinus pyralis （Ppy） catalyzes a two-step reaction that results in the oxidation of D-luciferin accompanied by emission of yellow?green light with a peak at 560 nm. However, some biological applications are limited by the low stability of the luciferase, so we combined amino acid mutations to enhance the enzyme’s thermostability and was eventually named Luciferase Ⅱ.