Objective: To assess the clinical application value of the combined localization of X-ray mammography and ultrasonography in biopsy of breast microcalcification. Methods: Under X-ray mammography, three-dimensional lesion localization was performed with the placement of a double-hook localization needle that was fixedly positioned after it reached the lesion; a skin marker line was drawn after detection of the lesion site through searching the double-hook needle with ultrasonography, and a single-hook localization needle was inserted; a 3-cm incision was made over the skin marker line, the mammary tissue bearing lesions and the single-hook needle were exposed and fixed by using special retractor, and then the lesion region was excised by using a rotary cutting tool; the cylindrical specimen was placed on a scaled specimen holder to take radiographic images for verifying the complete removal of the calcification lesions and ascertaining the three-dimensional location of the lesions in the specimen so as to facilitate the following pathological works. Results: A total of 108 patients with breast microcalcification that was classified as BI-RADS 4A underwent above procedures. The average distance of the lesion from the double hook needle was 4.1 mm (less than those by conventional methods), the average weight of specimen was 8.5 g (lighter than those by conventional methods), and all lesions were precisely excised by one session. Of the 108 patients, 20 cases had positive lesions that included atypical ductal hyperplasia in 7 cases, ductal carcinoma in situ of breast in 7 cases, ductal carcinoma in situ with focal invasive carcinoma in 3 cases, and invasive ductal carcinoma in 3 cases, and 88 cases had negative lesions. Both location and shape of the microcalcification lesion showed no significant relation with the detection of breast cancer (both P>0.05). Conclusion: Combined localization of X-ray mammography and ultrasonography has the advantages of accurate lesion localization, proper biopsy method, and small volume of specimen, as well as providing precise position of lesions in the specimen for pathological examination. Further, it does not exert any influence on the breast appearance in patients with benign lesions.