Nucleic acid nanomaterials with good biocompatibility, biodegradability, and programmability have important applications in biomedical field. Nucleic acid nanomaterials are usually combined with some inorganic nanomaterials to improve their biological stability. However, undefined toxic side effects of composite nanocarriers hamper their application in vivo. As a nanotool capable of avoiding potential biotoxicity, nanostructures composed entirely of DNA oligonucleotides have been rapidly developed in the field of biomedicine in recent years. Rolling circle amplification (RCA) is an isothermal enzymatic nucleic acid amplification technology for large-scale production of periodic DNA/RNA with pre-designed desirable structures and functions. RCA products with different functional parts can be customized by changing the sequence of the circular template, thereby generating complex multifunctional DNA nanostructures, such as DNA nanowire, nanoflower, origami, nanotube, nanoribbon, etc. More importantly, RCA products as nonnicked building blocks can enhance the biostability of DNA nanostructures, especially in vivo. These RCA products-based nucleic acid nanostructures can be used as scaffolds or nanocarriers to interact or load with metal nanoparticles, proteins, lipids, cationic polymers, therapeutic nucleic acids or drugs, etc. This paper reviews the assembly strategies of RCA based DNA nanostructures with different shape and their applications in biosensing, bioimaging and biomedicine. Finally, the development prospects of the nucleic acid nanomaterials in clinical diagnosis and treatment of diseases are described. STATEMENT OF SIGNIFICANCE: As a nanotool capable of avoiding potential biotoxicity, nanostructures composed entirely of DNA oligonucleotides have been rapidly developed in the field of biomedicine in recent years. Rolling circle amplification (RCA) is an isothermal enzymatic nucleic acid amplification technology for large-scale production of periodic DNA/RNA with pre-designed desirable structures and functions. This paper reviews the construction of various shapes of pure nucleic acid nanomaterials based on RCA products and their applications in biosensing, bioimaging and biomedicine. This will promote the development of biocompatible DNA nanovehicles and their further application in living systems, including bioimaging, molecular detection, disease diagnosis and drug delivery, finally producing a significant impact in the field of nanotechnology and nanomedicine.