Abstract: Circular RNAs (circRNAs), single-stranded covalently closed RNA molecules characterized by unique topological structures, have emerged as pivotal tools in biomedical research due to their exceptional biostability, low immunogenicity, and therapeutic versatility. This review briefly introduces the biological functions of endogenous circRNA and discusses their expanding applications in drug development, with a focus on the latest trends in the development of circRNA aptamers, circRNA sponge, guide circRNA, circRNA-based vaccines and circRNA-based CAR-T immunotherapies. Besides, it comprehensively analyzes the in vitro synthesizing methodologies, including chemical and enzymatic strategies, and analyzes in depth the challenges in circRNA drug development, The review concludes by exploring future directions for circRNA technology and its transformative potential in advancing the development of vaccines and therapeutics.