In vivo fiber photometry is a powerful technique to analyze the dynamics of population neurons during functional study of neuroscience. Here, we introduced a detailed protocol for fiber photometry-based calcium recording in freely moving mice, covering from virus injection, fiber stub insertion, optogenetical stimulation to data procurement and analysis. Furthermore, we applied this protocol to explore neuronal activity of mice lateral-posterior (LP) thalamic nucleus in response to optogenetical stimulation of primary visual cortex (V1) neurons, and explore axon clusters activity of optogenetically evoked V1 neurons. Final confirmation of virus-based protein expression in V1 and precise fiber insertion indicated that the surgery procedure of this protocol is reliable for functional calcium recording. The scripts for data analysis and some tips in our protocol are provided in details. Together, this protocol is simple, low-cost, and effective for neuronal activity detection by fiber photometry, which will help neuroscience researchers to carry out functional and behavioral study in vivo.