Objective: To investigate the clinical application of fluorescence imaging technology combined with methylene blue tracer in sentinel lymph node biopsy for breast cancer. Methods: A total of 150 patients with breast cancer were selected in this study. Of the patients, 50 cases underwent sentinel lymph node biopsy (SLNB) with indocyanine green (ICG) combined with methylene blue (the combination group), 50 cases underwent SLNB with ICG alone (the ICG group), while 50 cases underwent SLNB with methylene blue alone (the methylene blue group); intraoperative frozen section was performed, followed by conventional histopathology. Results: In the combination group, patients developing lymphatic vessels was 94% (47/50), and of the remaining three cases, two patients developed lymph node incision, lymph fluoroscopic visualization was 92% (46/50). The total detection rate of sentinel lymph nodes (SLNs) in the combination group was 98% (49/50) and the number of detected SLNs was 180, including 10 positive patients (20.41%); the total detection rate in the ICG group was 90% (45/50) and the detection number was 158, of which 8 patients was positive (17.78%); the total detection rate in the methylene blue group was 88% (44/50) and its detected number was 150, including 7 positive patients (15.91%); the related indicators in the combination group were higher than that in the ICG group or the methylene blue group. Conclusion: Compared to a single injection of ICG or methylene blue, ICG combined with methylene blue tracer method can significantly improve the detection rate and the number of the sentinel lymph node in breast cancer patients, which could provide an accurate guide on the selection of programs for breast cancer surgery.