Janus nanoparticles refer to a kind of asymmetric-structured nanoparticles composed of two or more distinct sides with differences in chemical nature and/or polarity on each side and thus can integrate two or more properties in one single particle. Due to their unique structure and surface properties, Janus nanoparticles have shown broad application potentials in optics, nuclear magnetic resonance, multi-mode imaging, and other fields. Unlike traditional contrast agents used in biological imaging, Janus nanoparticles are asymmetrically and directionally oriented to ensure stable partitioning of individual nanoparticles while integrating more functions. Much advancement have been carried out in the past few years, with some studies partially covering bioimaging applications. However, to our best knowledge, there are still no review papers specifically dedicated to the bioimaging applications with Janus nanoparticles. Bearing this in mind and taking the current challenges in this field into consideration, herein, we discuss representative approaches orchestrated for bioimaging applications, with the focus on the improvement of imaging quality brought by Janus nanoparticles and the development of multifunctional nanoplatforms in biological imaging fields, such as theranostics and therapies. Finally, based on the research experience of our group in this field, prospects for future research trends are put forward to provide new ideas for designing new Janus nanoparticles for clinical bioimaging.