The optical trapping, sensing, and imaging of nanostructures and biological samples are research hotspots in the fields of biomedicine and nanophotonics. However, because of the diffraction limit of light, traditional optical tweezers and microscopy are difficult to use to trap and observe objects smaller than 200 nm. Near-field scanning probes, metamaterial superlenses, and photonic crystals have been designed to overcome the diffraction limit, and thus are used for nanoscale optical trapping, sensing, and imaging. Additionally, photonic nanojets that are simply generated by dielectric microspheres can break the diffraction limit and enhance optical forces, detection signals, and imaging resolution. In this review, we summarize the current types of microsphere lenses, as well as their principles and applications in nano-optical trapping, signal enhancement, and super-resolution imaging, with particular attention paid to research progress in photonic nanojets for the trapping, sensing, and imaging of biological cells and tissues.