Objective To investigate the feasibility, safety, and clinical application value of single photon emission computed tomography/computed tomography (SPECT/CT)-guided bone marrow biopsy (BMB) in breast cancer (BC) patients with suspected bone metastases (BM) and compare its diagnostic performance for detection of BM with SPECT/CT. Methods The records of breast cancer patients referred for bone scintigraphy (BS), SPECT/CT and SPECT/CT-guided BMB from January of 2018 to June of 2021 in our hospital were retrospectively reviewed. 49 Patients were consecutively included in this study, all 49 specimens were analyzed by pathological and immunohistochemical studies.The biopsy success rate, total examination time, biopsy operation time, complications, CT radiation dose, and pathological and immunohistochemical results were recorded. The diagnostic performance based on SPECT/CT and SPECT/CT-guided BMB were compared with pathological, immunohistochemical examinations and the results of subsequent follow-up. Results Bone samples of the sites with high uptake were obtained in all 49 patients under BMB. No severe postoperative complications occurred. Among all 49 cases, 34 specimens were positive for metastatic breast cancer (69%, 34/49), and positive for benign tissue in 15 cases (31%, 15/49). 1 case of 15 cases was subsequently diagnosed as metastatic breast cancer according to the follow-up result. SPECT/CT-guided BMB demonstrated significantly higher negative predictive value (NPV) when compared to SPECT/CT (p = 0.021 < 0.05). Patients with differential expression of ER, PR, and HER-2 between primary lesions and metastatic lesions accounted for 12, 17, and 5 cases, respectively, and the changing rates were 35.2% (12/34), 50% (17/34), and 14.7% (5/34), respectively. Molecular subtype changes occurred in 7 patients, accounting for 47% (16/34) of metastatic patients. Conclusion It is insufficient to evaluate BM in BC patients using SPECT/CT imaging. SPECT/CT-guided BMB provided significantly higher sensitivity and NPV than SPECT/CT for detection of BM in BC patients. Our research redefines a new approach which can confirm diagnosis and potential molecular subtype changes for suspected bone metastatic lesions in BC patients, which can offer important opportunities for precision treatment and improved quality of life of BC patients with BM.