DNA-based materials exhibit great potential in biomedical applications due to their excellent sequence programmability and unique functional designability. Rolling circle amplification (RCA) is an efficient isothermal enzymatic amplification strategy to produce ultralong single-stranded DNA with customized functional moieties. The rational design of RCA templates and the introduction of other functional components enable RCA-based DNA materials with structural dynamic responsiveness and diverse biological functions, facilitating the development of DNA-based materials in biomedical applications. In this review, the principle and synthetic methods of RCA and the recent progress of RCA-based DNA nanostructures for therapeutics and bioimaging are summarized and discussed. The future challenges and opportunities for RCA-based DNA nanostructures are discussed at the end of the study. We envision that the development of RCA-based DNA nanostructures will provide more possibilities for precision medicine.