The challenge of "false positive" signals significantly complicates tumor localization and surgical resection, which are pivotal for successful tumor surgeries. Therefore, the development of a method for preoperative tumor localization and intraoperative margin determination holds considerable promise for improving surgical outcomes. In this study, a zero-crosstalk ratiometric tumor-targeting near-infrared (NIR) fluorescent probe was developed for precise cancer diagnosis and intraoperative navigation via NIR fluorescence imaging. This probe integrates a tumor-targeting moiety that selectively homes in on hepatocellular carcinoma cells and exhibits a highly sensitive ratiometric response to γ-glutamyltranspeptidase (GGT), characterized by a substantial emission shift from 830 to 650 nm. This unique NIR ratiometric emission property significantly enhances its effectiveness in early diagnosis and imaging-guided surgery resection. Furthermore, this zero-crosstalk probe successfully monitors GGT activity in blood, cells, and in vivo, endowing its potential for early cancer diagnosis in the clinic. Due to its efficient targeting and sensitive in situ response to GGT in both subcutaneous tumors and orthotopic hepatic tumors, the probe exhibits accurate detection capability for hepatic tumors. Additionally, by leveraging zero-crosstalk ratiometric NIR fluorescence imaging, this tumor-targeting probe can serve as a sprayable tool for delineating tumor margins with precision, thereby furnishing real-time intraoperative imaging guidance during tumor resection surgery.