The mono-julolidine-fused analog of SiR (JSiR) is widely used as a NIR fluorophore for biological imaging. In this study, we therefore examined JSiR as the scaffold fluorophore of a NIR fluorescence probe for hypoxia. We also evaluated the responsiveness of the synthesized probe, T-azoJSiR640, to oxygen concentration and confirmed its utility to selectively detect severe hypoxia in vivo.