Fluorescence molecular tomography (FMT) could exploit the distribution of fluorescent biomarkers that target tumors accurately and effectively, which enables noninvasive real-time 3-D visualization as well as quantitative analysis of small tumors in small animal studies in vivo. Due to the difficulties of reconstruction, continuous efforts are being made to find more practical and efficient approaches to accurately obtain the characteristics of fluorescent regions inside biological tissues. In this paper, we propose a region reconstruction method for FMT, which is defined as an L1-norm regularization piecewise constant level set approach. The proposed approach adopts a priori information including the sparsity of the fluorescent sources and the fluorescent contrast between the target and background. When the contrast of different fluorescent sources is low to a certain degree, our approach can simultaneously solve the detection and characterization problems for the reconstruction of FMT. To evaluate the performance of the region reconstruction method, numerical phantom experiments and in vivo bead-implanted mouse experiments were performed. The results suggested that the proposed region reconstruction method was able to reconstruct the features of the fluorescent regions accurately and effectively, and the proposed method was able to be feasibly adopted in in vivo application.