Perineural invasion (PNI) is the process of neoplastic invasion of nerves. The nerve sheath includes three layers, that is, the epineurium, perineurium and endoneurium from the outside to the inside. Any one of them is invaded by malignant tumor cells, or more than 33% of the circumference of the nerve should be surrounded by tumor cells, then it can be defined as perineural invasion (PNI). Perineural invasion is another way of tumor cell metastasis, which not only indicates the poor prognosis of patients but also indicates the decline of survival rate. In this study, multiphoton microscopy (MPM) which is based on the intrinsic nonlinear optical signals—two-photon excited fluorescence (TPEF) and second harmonic generation (SHG) was applied to identify the perineural invasion in breast cancer patients, including the recognition of normal nerves and tumor cells infiltrating into the nerve. Our aim is to determine the potential feasibility of using this microscopy to identify tumor invasion around the nerves. The results mean that it is feasible to detect perineural invasion with multiphoton microscopy, and to provide a new auxiliary tool for pathologists to quickly and effectively diagnose perineural invasion.