Objective To evaluate intraoperative tracing of sentinel lymph node(SLN) by fluorescence staining combined with dye dyeing.Methods A total of 174 patients with breast cancer were randomly divided into three groups : the group A with 57 patients receiving methylene blue (MB), the group B with 58 patients receiving indocyanine green(ICG) as the lymphatic mapping tracers,the group C with 59 patients receiving MB and ICG.The sentinel and axillary lymph node of level Ⅱ, Ⅲ was excised, followed by conventional histopathology.Results There was no significant difference among the three groups in the term of visualized detection rate (x2=2.96, P =0.241).There was statistical significant difference among three groups in the term of detected lymph nodes(F=15.34, P＜0.05).Comparing with the three groups, the number of detected lymph nodes of A and B group had no significant differences(P=0.07) ,the number of detected lymph nodes of C was higher than that of A and B group, and the difference was significant(P＜0.05).There was statistical significant difference among three groups in the term of SLN positive rate (x2 =6.75, P =0.039), and there was no significant difference among A and B group(P=0.915) ,SLN positive rate of C group was higher than than A and B group, and the difference was statistical significant (P＜0.05).Conclusion Intraoperative tracing of SLN by fluorescence staining combined with dye dyeing has the skin and subcutaneous reveal advantage.The use of ICG fluorescence and MB increases lymph node detection rate.