Tumor microenvironment (TME)-activatable probes have been proven to effectively increase signal-to-background ratios (SBRs) and improve the success rate of complete tumor resection. However, many fluorescence probes have to be loaded into a nanocarrier for tumor targeted delivery, which consequently encounters poor drug loading, heterogeneous composition and non-encapsulated drug aggregates occurred during nanoformulation fabrications. Herein, a nitroreductase (NTR)-activated “OFF-ON” near-infrared fluorescence nanoprobe, named NanoBodipy, was synthesized by the spontaneous self-assembling of NTR-responsive dye-polyethylene glycol (PEG) amphiphilic polymer in water. The NTR-responsive dye acted as the hydrophobic segment in the amphiphilic polymer, yielding a homogeneous composition and a high loading of 12.2 wt% (according to calculation) in the synthesized NanoBodipy. The synthesized NanoBodipy can efficiently accumulate in tumors via the enhanced permeability and retention (EPR) effect, enabling non-invasive tumor-targeted fluorescence imaging and guiding complete tumor resection. Once the synthesized NanoBodipy entered the tumor cells, they dissociated and were activated by overexpressed NTR. With the real-time fluorescence guide of NanoBodipy, complete tumor resection surgery was performed successfully.