Abstract The development of minimally invasive surgery has greatly advanced precision tumor surgery, but sometime suffers from restricted visualization of the surgical field, especially during the removal of abdominal tumors. A 3‐D inspection of tumors could be achieved by intravenously injecting tumor‐selective fluorescent probes, whereas most of which are unable to instantly distinguish tumors via in situ spraying, which is urgently needed in the process of surgery in a convenient manner. In this study, this work has designed an injectable and sprayable fluorescent nanoprobe, termed Poly‐g‐BAT, to realize rapid tumor imaging in freshly dissected human colorectal tumors and animal models. Mechanistically, the incorporation of γ ‐glutamyl group facilitates the rapid internalization of Poly‐g‐BAT, and these internalized nanoprobes can be subsequently activated by intracellular NAD(P)H: quinone oxidoreductase‐1 to release near‐infrared fluorophores. As a result, Poly‐g‐BAT can achieve a superior tumor‐to‐normal ratio (TNR) up to 12.3 and enable a fast visualization (3 min after in situ spraying) of tumor boundaries in the xenograft tumor models, Apc min/+ mice models and fresh human tumor tissues. In addition, Poly‐g‐BAT is capable of identifying minimal premalignant lesions via intravenous injection.