Background/Objectives Nanobodies are the smallest biologic antigen‐binding fragments derived from camelid‐derived antibodies. Nanobodies effect a peak tumor signal within minutes of injection and present a novel opportunity for fluorescence‐guided surgery (FGS). The present study demonstrates the efficacy of an anti‐CEA nanobody conjugated to near‐infrared fluorophore LICOR‐IRDye800CW for rapid intraoperative tumor labeling of colon cancer. Methods LS174T human colon cancer cells or fragments of patient‐derived colon cancer were implanted subcutaneously or orthotopically in nude mice. Anti‐CEA nanobodies were conjugated with IRDye800CW and 1–3 nmol were injected intravenously. Mice were serially imaged over time. Peak fluorescence signal and tumor‐to‐background ratio (TBR) were recorded. Results Colon cancer tumors were detectable using fluorescent anti‐CEA nanobody within 5 min of injection at all three doses. Maximal fluorescence intensity was observed within 15 min–3 h for all three doses with TBR values ranging from 1.3 to 2.3. In the patient‐derived model of colon cancer, fluorescence was detectable with a TBR of 4.6 at 3 h. Conclusions Fluorescent anti‐CEA nanobodies rapidly and specifically labeled colon cancer in cell‐line‐based and patient‐derived orthotopic xenograft (PDOX) models. The kinetics of nanobodies allow for same day administration and imaging. Anti‐CEA‐nb‐800 is a promising and practical molecule for FGS of colon cancer.