Intraoperative surgical margin assessment remains a challenge during breast-conserving surgery. Here, we report a combined strategy of immuno–positron emission tomography (PET) for preoperative detection of breast cancer and guided assessment of margins in breast-conserving surgery through second near-infrared (NIR-II) fluorescence imaging of trophoblastic cell surface antigen 2 (TROP2). We demonstrated that the intensity of PET signals in the tumors was nearly five times higher than in normal breast tissue with a zirconium-89 tracer conjugated to sacituzumab govitecan (SG) in a mouse spontaneous breast cancer model, enabling the identification of tumors. We further generated a NIR-II probe of indocyanine green conjugated to SG (ICG-SG) and developed a rapid incubation imaging method for intraoperative margin assessment in a relevant time window for the operation workflow. The ICG-SG NIR-II fluorescence image guidance was first verified to remove tumors completely and accurately in mouse breast cancer models. Moreover, the rapid incubation imaging method was applied to distinguish benign and malignant breast lesions in samples from 26 patients with breast cancer. Therefore, we have developed both nuclide and optical probes targeting TROP2 for rapid and precise identification of tumor margins during breast-conserving surgery in humans.