A non-contact Fluorescence Diffuse Optical Tomography (FDOT) system based Electron-Multiplying CCD(EMCCD) was setup for in vivo imaging of mice. A novel Data-Extraction-Extension-Method (DEEM) was investigated to avoid the complex focus correction calculation for the optical lens and alleviate the ill-posedness of the inverse problem. Accordingly, a non-contact FDOT image reconstruction method based on the DEEM was developed. A lot of FDOT experiments were carried on the solid phantoms, which proved that the best reconstruction results can be achieved when the Data-Extraction-area is half of the whole projection of the cylinder phantom on the CCD and is decomposed into three sub-areas. To further validate the method, the in vivo mouse with fluorescence target embedded was fabricated and the FDOT experiment was carried on. Reconstructed images from FDOT system and those from the Micro-CT demonstrate that the combination of the non-contact system and the proposed reconstruction method based on the DEEM can obtain the location and yield of the fluorescence target in the mouse correctly. The system can be further developed to detect the subcutaneous tumor of the small animals.