A near-infrared fluorochrome, GPU-311, was designed, synthesized and evaluated for its application in non-invasive imaging of tumor hypoxia. Efficient synthesis was achieved by nucleophilic substitution and click chemistry ring using the bifunctional tetraethylene glycol linker 2 containing thiol and azide groups for the conjugation of the propargylated nitroimidazole 1 and the heptamethine cyanine dye 3 bearing a 2-chloro-1-cyclohexenyl ring. GPU-311 exhibited long excitation and emission wavelength (Ex/Em_785/802 nm) and a decent quantum yield (0.05). The water solubility and hydrophilicity of GPU-311 increased. After in-vitro treatment of SUIT-2/HRE-Luc pancreatic cancer cells with GPU-311, a higher level of fluorescence was observed selectively in hypoxia than in normoxia. However, in-vivo fluorescence imaging of a mouse xenograft model after GPU-311 administration revealed inadequate accumulation of GPU-311 in tumors due to its rapid elimination through the liver.