ObjectiveThe aim of this study was to determine the possibility of endoscopic sentinel lymph node biopsy of the head and neck region using indocyanine green‐neomannosyl human serum albumin (ICG:MSA) and a custom‐made intraoperative color‐and‐fluorescence‐merged imaging system (ICFIS).MethodsUsing mouse and rabbit models of tongue cancer, we performed sentinel lymph node biopsy using an ICG:MSA tracer and custom imaging system equipped with an endoscope.ResultsICG:MSA was localized to the cervical lymph node on the ipsilateral side for up to 30 minutes compared to ICG tracer (ICG n = 3, IGC:MSA n = 3). Detection of sentinel lymph nodes was attempted after injecting ICG:MSA in the mouse tongue cancer model (n = 3). During the operation, lymph nodes were easily identified and resected using ICFIS without the aid of other magnifiers. In the rabbit tongue cancer model (n = 3), the ICFIS equipped with 30‐degree endoscopy was used to confirm the feasibility of endoscopic sentinel lymph node biopsy. The entire sentinel lymph node biopsy procedure was performed using the ICFIS with the 30‐degree endoscope. We detected and dissected sentinel lymph nodes in the rabbit model without the other sentinel lymph node detection tools.ConclusionWe confirmed the usefulness of sentinel lymph node biopsy using a near infrared fluorescence technique and endoscopic system. Avoidance of radiation exposure and shine‐through phenomena, which are problems of sentinel lymph node biopsy using conventional radioisotopes, are advantages of our surgical technique. By combining the endoscopic system with a sentinel lymph node biopsy procedure, we avoided visible neck scars, which lead to excellent cosmetic outcomes.Level of EvidenceNA. Laryngoscope, 128:E135–E140, 2018