Reductase expression is a potential indicator of cellular pathology. Single-detection systems for reductases have been developed, however, the development of dual-detection systems remain largely unexplored. We rationally designed a dual-lock fluorescent probe that exhibited a high signal-to-noise ratio with a fluorescence Off-On response exclusively for the simultaneous activity of two reductases, NTR and hNQO1, which are overexpressed in cancer hypoxia. The system comprised a naphthalimide fluorophore with dual-lock control mediated by PET and ICT, a trimethyl-locked quinone group sensitive to hNQO1, and a nitrobenzyl carbamate group sensitive to NTR. This study employed a hypoxia model in HeLa cells to demonstrate that our developed dual-lock system detected hypoxia more effectively than single-detection systems. Moreover, it enabled noninvasive real-time monitoring of hypoxia in zebrafish embryos. Consequently, the dual-lock fluorescent probe, which strategically provides a fluorescence response only when both NTR and NQO1 are active, offers a novel diagnostic platform for both in vitro and in vivo applications, effectively detecting hypoxia and monitoring various pathological states.