Objective To investigate and analyze the difference of accuracy between preoperative puncture biopsy and intraoperative frozen pathology in the diagnosis of false negative for sentinel lymph nodes in breast cancer. Methods Retrospective analysis was performed on 116 patients. According to the pathological results, the pathological consistency between the observation group and the control group was statistically analyzed. Results There was no statistically significant difference in the consistent rate of different pathological age groups and tumor size groups between the preoperative biopsy group and the intraoperative frozen group (P > 0.05). The consistency rate of preoperative biopsy group was higher than that of intraoperative frozen group (P < 0.05). The false-negative rate was lower in preoperative biopsy group than in intraoperative frozen group (P < 0.05). Conclusion Preoperative puncture biopsy is more accurate than intraoperative frozen pathology in the diagnosis, and has guiding value for the optimization of treatment scheme.