Fiber photometry is a powerful technique to monitor neural activity dynamics of a particular population of neurons in living animal brains for the functional study. With the help of retrograde viral-based tracing, a specific neural circuit can be labeled with genetically encoded calcium indicators (GECIs) to facilitate in vivo activity measurement based on calcium imaging via fiber photometry. Here, we provided a simple and feasible protocol for fiber photometry of virus labeled specific neural circuitry in mice. The detailed protocol included virus injection, optic fiber implantation, Ca2+ signal detection, histology, imaging, and data analysis. Furthermore, we applied this protocol to label paraventricular thalamus (PVT) projection neurons targeting to the paraventricular nucleus of the hypothalamus (PVN), to specifically monitor neural activity of PVT–PVN circuit. Finally, we discussed the key points of the protocol, which should be concerned during fiber photometry of a specific neural circuit. In summary, this protocol is highly efficient and feasible and may contribute to the functional study of neural circuits.