The invention relates to a culture method of breast cancer organoids and a co-culture method of tumor-related fibroblasts. According to the invention, a more efficient and convenient improved system for key technical links such as PDO culture, passage, maintenance and the like of tumor tissue organoids of breast cancer patients is established, operation steps and methods for establishing and passage of a PDO cell line are simplified in multiple aspects, components of a culture medium, digestive juice, a washing solution and the like are optimized, and finally, a breast cancer PDO culture system is successfully established. Compared with the prior art, the culture method has the advantages that the success rate and the passage frequency are basically consistent, but the operation is simpler, the required time is shorter, a good foundation is laid for a PDO model to have better clinical and scientific research applications in the future, and the corresponding cost is saved. The inventionalso establishes a stable co-culture system of breast cancer PDO and tumor-associated fibroblasts (CAFs), simultaneous culture, amplification and passage of PDO and CAFs can be realized, and the PDOand the CAFs do not produce obvious influence on each other.