Abstract Introduction The postoperative survival of oral squamous cell carcinoma (SCC) relies on precise detection and complete resection of original tumors. The mucosal extension of the tumor is evaluated visually during surgery, but small and flat foci are difficult to detect. Real-time fluorescence imaging may improve detection of tumor margins. Materials and Methods In the current study, a peptide-based near-infrared (NIR) fluorescence dye, c-MET-binding peptide-indocyanine green (cMBP-ICG), which specifically targets tumor via c-MET binding, was synthetized. A prospective pilot clinical trial then was conducted with oral SCC patients and intraoperatively to assess the feasibility of cMBP-ICG used to detect tumors margins. Fluorescence was histologically correlated to determine sensitivity and specificity. Results The immunohistochemistry (IHC) results demonstrated increased c-Met expression in oral SCC compared with normal mucosa. Tumor-to-background ratios ranged from 2.71 ± 0.7 to 3.11 ± 1.2 in different concentration groups. From 10 patients with oral SCC, 60 specimens were collected from tumor margins. The sensitivity and specificity of discriminative value derived from cMBP-ICG application in humans were respectively 100% and 75%. Conclusions Topical application of cMBP-ICG is feasible and safe for optimizing intraoperative visualization and tumor margin detection in oral SCC patients, which could clinically increase the probability of complete resections and improve oncologic outcomes.