Fluorescence molecular tomography (FMT) supports monitoring molecular events non-invasively in vivo over a span of long time, and meets the demands of monitoring a process of life. Priori information can be applied to speed the complex and time-consuming reconstruction process and to enhance the quality of the reconstructed images for FMT. A method was proposed in the present paper to estimate rapidly the depth of fluorescence source. The estimation process was accomplished with an optimization algorithm, particle swarm optimization (PSO). Firstly the fluorescence intensity ratio Rf of two positions on the boundary of tissue was derived under extrapolated boundary condition and a diffusion model for the propagation of near-infrared photons in biological tissue. Then a PSO algorithm was applied to minimize the difference between the theoretical ratio RTf and the measured ratio RMf. The depth of fluorescence source was estimated after the rapid PSO optimization process. Two phantoms indicated that the proposed method can estimate the depth of single fluorescence source rapidly and easily without the time-consuming mesh generation and reconstruction process.