Fluorescence molecular tomography (FMT), as a promising technique for early tumor detection, can non‐invasively visualize the distribution of fluorescent marker probe three‐dimensionally. However, FMT reconstruction is a severely ill‐posed problem, which remains an obstacle to wider application of FMT. In this paper, a two‐step reconstruction framework was proposed for FMT based on the energy statistical probability. First, the tissue structural information obtained from computed tomography (CT) is employed to associate the tissue optical parameters for rough solution in the global region. Then, according to the global‐region reconstruction results, the probability that the target belongs to each region can be calculated. The region with the highest probability is delineated as region of interest to realize accurate and fast source reconstruction. Numerical simulations and in vivo experiments were carried out to evaluate the effectiveness of the proposed framework. The encouraging results demonstrate the significant effectiveness and potential of our method for practical FMT applications.