Rolling circle replication (RCR), including rolling circle amplification (RCA) and rolling circle transcription (RCT), is an isothermal enzymatic reaction. Because of its high amplification efficiency, RCR is a powerful biosensing tool for detecting biomolecules. In recent years, RCR has also been extended to the field of bioimaging to better understand biological pathways. Furthermore, RCR provides a simple technique to design and generate DNA/RNA structures with unique advantages in delivering drugs and enhanced targeting ability. In this review, we introduce the fundamentals of RCR and describe the most recent advances in RCR-based detection methods and delivery vehicles for biosensing, bioimaging, and biomedicine. Finally, some challenges and further opportunities of RCR-based biotechnology are discussed. Rolling circle replication (RCR) is an isothermal nucleic acid amplification approach that holds great potential in biotechnology.RCR can serve as a powerful biosensing platform for the detection of various biomolecules in bioanalysis and diagnosis.In bioimaging, RCR can provide a unique perspective in comprehensive understanding of biological processes and effective diagnosis of diseases.Programmable, biodegradable RCR-based assemblies have been used as versatile advanced drug carriers for targeted theranostics.