Purpose: Optical imaging is providing great advance and improvement in genetic and molecular imaging of animals and humans. Optical imaging system consists of optical imaging devices, which carry out major function for monitoring, tracing, and imaging in most of molecular in-vivo researches. In bio-luminescent imaging, small animals containing luciferase gene locally irradiate light, and emitted photons transmitted through skin of the small animals are imaged by using a high sensitive charged coupled device (CCD) camera. In this paper, we introduced optical imaging system for the image acquisition of bio-luminescent signals emitted from small animals. Materials and Methods: In the system, Nikon lens and four LED light sources were mounted at the inside of a dark box. A cooled CCD camera equipped with a control module was used. Results: We tested the performance of the optical imaging system using effendorf tube and light emitting bacteria which injected intravenously into CT26 tumor bearing nude mouse. The performance of implemented optical imaging system for bio-luminescence imaging was demonstrated and the feasibility of the system in small animal imaging application was proved. Conclusion: We anticipate this system could be a useful tool for the molecular imaging of small animals adaptable for various experimental conditions in future. (Nucl Med Mol Imaging 2009;43(4):344-351)