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 (Tm value) of 75.3 ± 0.3 °C. Additionally, the variant demonstrated efficient catalytic activity at moderate temperatures, exhibiting 3.8 and 3.7-fold higher catalytic efficiencies towards D-luciferin and ATP at 37 °C compared to Ppe146-1H2. Overall, Ppe146-LGR displayed mesophilic-like catalytic activity and thermophilic-like thermostability simultaneously. In addition to enhanced catalytic properties, Ppe146-LGR emitted longer-wavelength light (580 nm) and operated optimally at near-neutral pH, coordinating with the current demands of luciferase applications. Through validation via rapid bacterial detection and reporter gene assays, it has been demonstrated that Ppe146-LGR holds promise as a valuable tool in the field of bioluminescence technology.