Nucleic acid therapeutics, as a transformative modality in modern biopharmaceuticals, mediate disease treatment through precise gene expression modulation. Their remarkable therapeutic potential, such as in oncology, has been demonstrated clinically. The regulatory approval of nucleic acid drugs, exemplified by the SARS-CoV-2 mRNA vaccines, has not only validated their clinical translation potential, but also accelerated advancements in nucleic acid drug development platforms. The functional efficacy of nucleic acid drugs is fundamentally governed by their three-dimensional structural architecture, necessitating rigorous stability assessments throughout the development pipeline. Critical considerations include maintaining native conformation during delivery, achieving target-specific release profiles, and optimizing storage formulations for long-term stability. Circular dichroism (CD) spectroscopy has emerged as an indispensable analytical tool due to its exceptional sensitivity to chiral environments. CD enables precise detection of nucleic acid structural transitions, from base-stacking interactions to complex higher-order conformational changes, making it invaluable for structural characterization. This review summarizes recent research progress on the typical conformational characteristics of nucleic acid drugs and the factors influencing their stability. It focuses particularly on methodological advances in CD spectroscopy for structural analysis and quality control, providing critical insights to guide future research in this rapidly evolving field.