<p>Breast cancer remains a significant public health concern, and neoadjuvant therapy has significantly reconfigured the landscape of its clinical management. The intricate characteristics of the tumor microenvironment (TME) have profound implications for tumor development and therapeutic responses. Thus, understanding the dynamics of the TME during treatment is crucial. Advancements in molecular biology research, such as single-cell analysis and spatial omics technologies, provide valuable tools for investigating the complexities of the breast cancer microenvironment. These innovative approaches facilitate the identification of cellular heterogeneity, spatial interactions, and altered signaling pathways, illuminating the TME’s adaptive mechanisms in response to neoadjuvant therapy. In this review, we first elucidate the current status of neoadjuvant therapy in breast cancer. Subsequently, we outline cutting-edge molecular biology research methods and their applications within the breast cancer microenvironment. Finally, we provide an overview of the alterations in the components of the tumor microenvironment during neoadjuvant treatment for breast cancer, focusing on insights gained from single-cell analysis and spatial pathology.</p>