Catalytic hairpin assembly (CHA) appears to be a particularly appealing nucleic acid circuit because of its powerful amplification capability, simple protocols, and enzyme-free and isothermal conditions, and can combine with various signal output modes for the biosensing of various analytes. Especially in the last five years, vast CHA related studies have sprung up. With the deep exploration of the CHA mechanism, some novel and excellent CHA strategies have been proposed; meanwhile the CHA cascade strategies with various amplification techniques further improve the analysis performance. Furthermore, diverse CHA based biosensors have been tactfully engineered and extensively employed in imaging applications in living cells and in vivo ascribed to its gentle reaction, efficient amplification and universality. Hence, we present a comprehensive and systematic summary of the progress in CHA and its application in bioimaging and biomedicine to date. At first, we introduced the mechanism and diversification of CHA in detail, including the newly developed CHA and its ingenious combination with a variety of other technologies. Concurrently, we summarized the latest application progress of different CHA strategies in bioimaging and biomedicine, highlighting the merits and drawbacks of representative approaches. Finally, we put forward some views on the challenges and prospects of CHA in bioimaging and biomedicine in the future.