Colorectal cancer (CRC) remains the leading cause of cancer mortality worldwide despite therapeutic advances. Guanylyl cyclase C (GUCY2C), an intestinal epithelial receptor, is emerging as a promising diagnostic and therapeutic target for CRC. Thus, we are interested in developing a monoclonal antibody probe targeting GUCY2C for both in vitro and in vivo diagnosis and treatment of CRC. Methods: The GUCY2C-specific monoclonal antibody, PR15-7, was generated by hybridoma fusion. We developed [89Zr]Zr-DFO-PR15-7 for PET imaging, Cy5-PR15-7 for near-infrared fluorescence I (NIR-I) detection, and ICG-PR15-7 for NIR-II-guided surgical navigation. The therapeutic potential was evaluated using [177Lu]Lu-DOTA-PR15-7 for targeted radiotherapy. Biologic properties and antitumor activity of PR15-7 probes were evaluated in vitro and in vivo. Results: PR15-7 showed strong GUCY2C binding affinity and tumor selectivity. PR15-7 probes in antibody-based PET and NIR imaging revealed 3 times higher signal intensity in GUCY2C-positive tumors compared with controls. The NIR-II probe ICG-PR15-7 enabled precise intraoperative tumor visualization and complete resection in orthotopic models. Therapeutic administration of [177Lu]Lu-DOTA-PR15-7 significantly inhibited tumor growth, with standardized tumor volumes at 16 d being markedly smaller than those in the control groups. Conclusion: We have established both optical and radionuclide probes with PR15-7 as a versatile therapeutic strategy, providing valuable insights into targeted therapy for CRC.