Optical imaging of in vivo luminescence has been used extensively for the evaluation of biological events such as gene expression, protein-protein interaction, tumour growth, and metastasis in mice under anaesthesia. In most cases, the exposure time required for the CCD camera to take an image ranges from tens of seconds to more than 1 minute; therefore, these phenomena cannot be evaluated in real time. However, it would be more useful if these biological events could be detected in small animals at video rate or an equivalent time resolution. Here, we describe a protocol for luminescence imaging in a freely moving unshaved mouse using a bright version of a luminescent protein, Nano-lantern, and a system to capture both luminescence (dark-field) and whole animal (bright-field) images. The protocol includes not only the preparation of the mouse with luminescent tumour tissue inside the body but also how to set up the homemade luminescence imaging system, which includes an EM-CCD camera and an alternative shutter control devise in a dark box made from cardboard. This protocol could be applied for any luminescence imaging application in small organisms, including zebrafish, Drosophila, and Arabidopsis.