In this study, we used sugars as stabilizing additives to improve the thermostability and to inhibit aggregation of firefly luciferase. The combination of sucrose and trehalose has a strong stabilizing effect on firefly luciferase activity and prevents its thermoinactivation. These additives can also increase optimum temperature. It has been shown that the presence of both sucrose and trehalose can inhibit thermal aggregation of firefly luciferase and decrease bioluminescence decay rate. In order to understand the molecular mechanism of thermostabilization, we investigated the effects of sucrose and trehalose combination on the secondary structure of luciferase by Fourier transform infrared spectroscopy. Minor changes in content of secondary structure of firefly luciferase are observed upon treatment with additives.