Objective: To investigate the feasibility of using combined tracing method of indocyanine green (IGG) fluorescence and methylene blue dyeing in sentinel lymph node biopsy (SLNB) of breast cancer. Methods: Two hundred and seventy-six breast cancer patients admitted between March 2014 and October 2015 were selected. Of the patients, 131 cases underwent SLNB with ICG combined with methylene blue (combination group), while 145 cases underwent SLNB with methylene blue alone (methylene blue group); all cases underwent level I and II axillary lymph node dissection after SLNB. Results: The general clinical data had no statistical difference between the two group of patients (all P>0.05). In combination group, the detection rate of sentinel lymph nodes (SLNs) was significantly higher than that in methylene blue group (96.9% vs. 89.7%, P=0.017), the average number of detected SLNs was significantly greater than that in methylene blue group (3.0 vs. 2.1, P=0.011) and the false negative rate was lower than that in methylene blue group (7.1% vs. 10.9%), but the latter did not reach a statistical significance (P=0.813). Statistical analysis of the data of the whole group showed that the false negative rate between the two groups in cases with detected number of SLNs less than or equal to 2 was significantly higher than in those with detected number of SLNs equal to or more than 3(17.5% vs. 2.1%, P=0.033). Conclusion: Compared with methylene blue dyeing alone, the combined tracing of IGG fluorescence and methylene blue dyeing for SLNB in breast cancer has advantages of high detection rate and average number of detected SLNs as well as real-time lymphatic imaging. So it is recommended to be used under the circumstance of no conventional access for using radionuclide method.