Bioluminescence imaging (BLI) using engineered bioluminescent viruses has emerged as a powerful tool for real-time, noninvasive monitoring of viral replication in living animals. While traditional luciferase-based systems, such as firefly luciferase, have been widely used, the NanoLuc luciferase system offers distinct advantages, including its significantly smaller gene size, increased brightness, and independence from ATP as a cofactor, allowing for extracellular detection. However, the utility of NanoLuc has been limited by its traditional substrate, furimazine, which exhibits poor water solubility and potential cytotoxicity. In this study, we assessed fluorofurimazine (FFz), a novel substrate with improved water solubility and bioavailability, for tracking influenza A virus (IAV) replication in mice. Our findings demonstrate that FFz substantially enhances detection sensitivity in both respiratory organs and brain tissue without increasing toxicity, enabling more precise and sustained monitoring of IAV replication. In vitro, FFz generated higher photon flux at lower concentrations compared to furimazine, translating into superior in vivo sensitivity with reduced toxicity. Crucially, FFz did not alter the pathogenicity of IAV in mice, even at sublethal infectious doses, reinforcing its suitability for use in BLI-based viral pathogenicity studies. These results suggest that combining FFz with NanoLuc provides a more effective and less toxic approach for real-time tracking of viral infections in preclinical models.