Conjugated polymer dots or semiconducting polymer nanoparticles (Pdots) are nanoparticles prepared based on organic polymers. Pdots have the advantages of lower cost, a simple preparation process, good biocompatibility, excellent stability, and easy modification and regulation. Based on these characteristics, they can not only realize super-resolution imaging of subcellular structure, and the localization of deep tissue lesions, but also interact with various substrates to realize quantitative sensing. Besides the imaging and sensing application, these Pdots can also be used as a targeted therapeutic effective agent to achieve several biotherapy functions. In this review, we will focus on the applications of conjugated polymer dots, including related design and preparation strategies, as well as the current situation and future development of specific applications such as imaging and treatment.