Furin, an important member in the family of proprotein convertases, is a participant in the activation of various precursor proteins. The expression level of furin stays in a very low range in most normal cells, but elevates with a big margin in many cancer cells. More importantly, furin is closely related to tumor formation and migration. Herein, a furin-activatable near-infrared (NIR) fluorescent probe (HD-F) was first developed that allowed for specific, sensitive detection and imaging of furin both in vitro and in vivo. HD-F consists of a classical NIR fluorophore (HD), a furin-particular polypeptide sequence RVRR, and a self-eliminating linker. Without the interaction with furin, no noticeable fluorescence enhancement was detected, even over 3 days, demonstrating the excellent stability of HD-F. Upon conversion by furin, there was a distinct signal increase around 708 nm. It has achieved assay and visualization of endogenous furin in various cells, tumor tissues, and tumor-bearing mouse models. Importantly, HD-F is well-suited for monitoring the change of furin expression level in the process of hypoxia-inducible factor-1 stabilized by CoCl2. Moreover, HD-F could visualize the divergence in the expression level of furin between normal and cancer cells, indicating its potential in specific cancer imaging. Thus, this novel probe is able to serve as a potential tackle for better understanding of the intrinsic link between a hypoxic physiological environment and cellular carcinogenesis and predicting cancer in preclinical applications.