Luciferase, known for its exceptional catalytic bioluminescent properties, has been widely utilized in diverse applications within biotechnology and medical research. Currently, enhancing thermostability and catalytic activity is a primary focus for optimizing luciferase modifications to further expand its detection range and accuracy. This study revealed a highly thermostable luciferase variant from Photuris pennsylvanica, Ppe146-1H2, which inherently exhibits thermophilic enzyme characteristics that are not conducive for optimal catalytic performance in practical applications. Building upon structural analysis, this research engineered Ppe146-1H2 into Ppe146-LGR via the residue substitutions I422L, D435G, and I519R. Ppe146-LGR retained notably thermostability, exhibiting a melting temperature (T