PURPOSE: Breast cancer patients undergoing breast-conserving surgery may require a second operation if positive margins persist but current intraoperative methodologies often lack real-time and comprehensive assessments of tissue margins. This study addresses this critical gap by introducing a novel approach to enhance margin assessment in breast surgery. METHODS: A total of 252 fresh tissue blocks from 199 patients with different types of breast lesions were scanned with a customized swept-source optical coherence tomography (SS-OCT) system, and the OCT features of normal, benign, and malignant breast tissues, were systematically analyzed. RESULTS: The qualitative analysis results revealed that adipose tissue has high penetration depth and a typical honeycomb pattern, whereas fibrous tissue has the brightest grayscale values and a bundle-like structure. The lobular area appears as a dark region, and dilated ducts present a distinct tubular structure on B-scan images. Adenosis results in bright areas, fibroadenoma results in typical contour structures, phyllodes tumors present lobular structures, invasive carcinomas present a stellate pattern and low penetration depth, and mucinous carcinoma cancer cells are clearly visible within the low-scattering mucin. CONCLUSIONS: Importantly, we provide comparative OCT and hematoxylin and eosin (H&E) histology images for less common conditions, such as phyllodes tumors, intraductal papillomas, and mucinous carcinoma. For the first time, we established an 3D OCT-histopathology library with a large field of view and systematically analyzed the multidimensional features. This work strongly supports the feasibility of using OCT technology intraoperatively in surgery. Additionally, the OCT-histopathology library can help pathologists better understand and identify tissue features, thereby enhancing diagnostic efficiency.