Objective: Lingual lymph node (LLN) metastasis is regarded as an indicator of unfavorable prognosis and a crucial sign of the high degree of primary tumor aggressiveness. However, detecting LLN metastasis is an important but frequently overlooked aspect of diagnosis and surgical treatment planning. The study aims to identify LLNs by intraoperative near-infrared (NIR) fluorescence imaging with indocyanine green absorbed into human serum albumin (ICG: HSA) and describe the presence of lymphatic drainage channels from the floor of the mouth in patients with tongue carcinoma. Materials and Methods: 21 patients diagnosed with cT1-T4 squamous cell carcinoma (SCC) of the tongue margin and scheduled to undergo tumor resection and unilateral neck dissection were enrolled. After exposing the neck, the patients were injected with 0.3 ml of ICG: HSA (500 μM) in three quadrants around the tumor, excluding the mucous membrane of the basal region cavity. Employing a near-infrared fluorescence imaging system, the fluorescence of levels I, II, III, and IV was measured during neck dissection. Results: LLNs were detected in four patients and were identified as metastatic LLNs in all 21 patients. The near-infrared fluorescence imaging system showed the existence of lymphatic drainage channels in the floor of the mouth. In patients receiving peritumoral injection of ICG: HSA, the mean fluorescence intensity (MFI)of metastatic lymph nodes (LNs) (178.4 ± 64.39, mean ± SD) was higher than that in non-metastatic LNs (132.0 ± 76.5, mean ± SD) (p < 0.05). Conclusion: NIR fluorescence imaging with ICG: HSA could be used for intraoperative identification of LLNs and assist in the determination of metastatic lymph nodes for tongue carcinoma patients. Additionally, this finding demonstrates the feasibility of near-infrared fluorescence imaging in defining lymphatic drainage channels in the head and neck.