Brest cancer has historically ranked number one in the global incidence of malignant tumors in women. The latest International Agency for Research on Cancer by the World Health Organization shows that breast cancer has surpassed lung cancer as the leading type of cancer worldwide. Multiphoton microscopy (MPM) is an advanced medical imaging technique based on nonlinear optical effects, such as multiphoton excited fluorescence and harmonic generation, occurring owing to laser interactions with biological tissues. MPM offers the advantage of a resolution comparable to digital histopathology as well as requires no labeling or sectioning while providing three-dimensional optical imaging that reflects the metabolic function information of cells, three-dimensional targeted ablation capability, low photobleaching and photodamage, and a high imaging contrast. Therefore, MPM has garnered considerable research interest locally and globally in the field of basic and applied research in medical pathological diagnosis, particularly in the clinical translation of tumor pathological diagnosis. Herein, the application progress of MPM in the prognostic prediction of breast tumor microenvironments is introduced in detail and its future development prospects are presented.