Abstract: The sentinel lymph node ( SLN ) biopsy has become a standard surgical procedure for staging the breast cancer, instead of axillary lymph node dissection ( ALD ). Using an appropriate tracer can increase the success rate and shorten the learning curve, so that the SLN procedure can be made accessible in primary hospitals. This study aims to discuss a novel method for sentinel lymph node ( SLN ) identification by fluorescent imaging. Methods: The optimal excitation and emission spectrum of MB solution were determined using a fluorescence spectrometer. The MB solution was subcutaneously injected to the breast of rabbit. A fluorescence spectrum in vivo imaging system of small animals was used to observe the lymphatic drainage and axillary SLN uptake of MB. The blue-stained SLNs were searched in the armpit, and fluorescence property was observed. Results: MB has a property of auto-fluorescence. The near-infrared ( NIR ) fluorescence of MB played a role for labeling the in vivo axillary lymph node drainage and mapping SLN. Conclusion: NIR fluorescence reagent may be a better tracer in mapping SLN for its visibility in vivo and in vivo.