Soft fluorescent nanomaterials have attracted recent attention as imaging agents for biological applications, because they provide the advantages of good biocompatibility, high brightness, and easy biofunctionalization. Here, we provide a survey of recent developments in fluorescent soft nano-sized biological imaging agents. Various soft fluorescent nanoparticles (NPs) (including dye-doped polymer NPs, semiconducting polymer NPs, small-molecule organic NPs, nanogels, micelles, vesicles, and biomaterial-based NPs) are summarized from the perspectives of preparation methods, structure, optical properties, and surface functionalization. Based on both optical and functional properties of the nano-sized imaging agents, their applications are then reviewed in terms of in vitro imaging, in vivo imaging, and cellular-process imaging, by means of specific or nonspecific targeting.