Optical coherence tomography (OCT) is a well-developed technology that utilizes near-infrared light to reconstruct three-dimensional images of biological tissues with micrometer resolution. Improvements of the imaging contrast of the OCT technique are able to further widen its extensive biomedical applications. In this study, Janus microspheres were developed and used as a positive contrast agent for enhanced OCT imaging. Phantom and ex vivo liver tissue experiments as well as in vivo animal tests were conducted, which validated that Janus microspheres, as a novel type of OCT tracer, were very effective in improving the OCT imaging contrast.