Janus particles, which are named after the two-faced Roman god Janus, have two distinct sides with different surface features, structures, and compositions. This asymmetric structure enables the combination of different or even incompatible physical, chemical, and mechanical properties within a single particle. Much effort has been focused on the preparation of Janus particles with high homogeneity, tunable size and shape, combined functionalities, and scalability. With their unique features, Janus particles have attracted attention in a wide range of applications such as in optics, catalysis, and biomedicine. As a biomedical device, Janus particles offer opportunities to incorporate therapeutics, imaging, or sensing modalities in independent compartments of a single particle in a spatially controlled manner. This may result in synergistic actions of combined therapies and multi-level targeting not possible in isotropic systems. In this review, we summarize the latest advances in employing Janus particles as therapeutic delivery carriers, in vivo imaging probes, and biosensors. Challenges and future opportunities for these particles will also be discussed.