Objective: To explore the concentration range and penetration depth of methylene blue near-infrared fluorescence imaging, and to clarify the role of methylene blue in oral lymphatic drainage and sentinel lymph node localization, so as to lay a foundation for the potential research and application of sentinel lymph node in oral cancer. Methods: 10% (mass fraction) methylene blue injection was diluted into 29 different concentrations with 0.9% (mass fraction) normal saline, and the concentration range of methylene blue near-infrared fluorescence imaging was determined by near-infrared fluorescence imager. The maximum penetration depth of methylene blue near-infrared fluorescence was determined by covering pigskin with different thicknesses (1, 2, 3, 4 and 5 mm) in methylene blue solution. 0.2 mL methylene blue solution was injected into the submucosal 0.5 cm at the lateral margin of tongue on one side of the rats. The near-infrared fluorescence imager was used for continuously monitoring for 3 hours. The first near-infrared fluorescence hotspot was identified as sentinel lymph node and labeled by percutaneous observation. The rats were then sacrificed and dissected in the head and neck. Near-infrared fluorescence imaging was performed again to observe whether the fluorescent tissue was consistent with the labeled fluorescent hotspot in vitro, and the presence of lymphoid tissue was confirmed by pathological examination after resection. Results: Except that no fluorescence signals were detected in the blank control groups, the fluorescence intensity of methylene blue increased first and then decreased with its solution concentration decreased. When the concentration of methylene blue was diluted to the picomole level, the fluorescence signal could still be detected. The maximum penetration depth of methylene blue fluorescence was 4 mm. Methylene blue near-infrared fluorescence could be localized in oral lymphatic drainage and sentinel lymph node. The fluorescence was sustained for more than 3 hours after methylene blue injection. Methylene blue solution concentrations of 3.34 mmol/L, 6.68 mmol/L, 13.37 mmol/L and 26.74 mmol/L were selected in the rats to map sentinel lymph node by near-infrared fluorescence. Conclusion: Methylene blue near-infrared fluorescence has a certain penetrating ability and can transcuta-neously map the sentinel lymph node and their associated lymphatic vessels in rats, which is expected to be further applied in the study of sentinel lymph node in oral cancer.