Bioluminescence imaging (BLI) enables in vivo imaging of molecular and cellular processes. It has gained in popularity over the past decade because of its easy translation from in vitro to in vivo experiments, its sensitivity, and its ease of use. However, experience in applying BLI in living subjects is still limited, and many researchers have encountered unexpected or biased BLI readout and reported important influencing factors. In this review, we summarize both the biological and physical effects that occur at the enzyme level or during light propagation towards the camera. The knowledge and detection of such factors, together with the development of new strategies and better BLI compounds, will improve the accuracy of the technique in the future.