Objective: To investigate the clinical application value of 99Tcm-colloidal rhenium sulfide in sentinel lymph nodes (SLNs) biopsy in patients with breast cancer. Methods: Thirty patients with T1–T2 breast cancer were enrolled. Patients were injected with 99Tcm-colloidal rhenium sulfide under the areola 15–24 h before operation, and lymphoscintigraphy and mapping of the SLNs was subsequently performed with SPECT/CT. Gamma probe-guided SLNs detection and biopsy was employed during surgery before the breast cancer resection. The results of SLN biopsy and histological examination were compared and analyzed after surgery. Results: SLNs were detected in 29 of the 30 patients and a total of 31 SLNs were identified. The detection rate and accuracy rate was 97.67% and 100%, respectively. The coincidence rate of the SLNs status and axillary lymph node metastasis was 100%, and no false positive or negative detections occurred. Conclusion: 99Tcm-colloidal rhenium sulfide is an ideal tracer for SLNs biopsy in patients with breast cancer.