Reduction-sensitive polymeric nanocarrier with near-infrared fluorescence probe has been prepared. Disulfide-cross-linked polypeptide nanogel with near-infrared fluorescence property (NIRF nanogel) was first synthesized, then the anticancer drug doxorubicin was encapsulated into polypeptide core of the NIRF nanogel to prepare a drug carrier with near-infrared fluorescence (NIRF prodrug). In vitro drug release study of the NIRF prodrug revealed an accelerated release behavior in the presence of 10 mM glutathione (GSH). Cellular uptake studies of both the NIRF nanogel and NIRF prodrug showed that they could enter cell via endocytosis. With the aid of NIRF labeling, direct imaging of the drug release from NIRF nanogel was accomplished, and drug molecules released subsequently migrated into nucleus while the NIRF nanogel still remained in cytoplasm. In vivo distribution of the NIRF nanogel and NIRF prodrug on tumor-bearing nude mice shows that both of them accumulated at tumor place at 24 h after tail veil injection via enhanced permeability and retention (EPR) effect. The NIRF prodrug prepared here has the potential application for the theranosis of cancer.