With the growing interest in nucleic acids and nanozymes, nucleic acid nanozymes (NANs) have emerged as a promising alternative to traditional enzyme catalysts, combining the advantages of nucleic acids and nanomaterials, and are widely applied in the field of biosensing. This review provides a comprehensive overview of recent studies on NAN-based biosensors. It classifies NANs based on six distinct enzymatic activities: peroxidase-like, oxidase-like, catalase-like, superoxide dismutase-like, laccase-like, and glucose oxidase-like. This review emphasizes how the catalytic activity of nanozymes is significantly influenced by the properties of nucleic acids and explores the regulatory mechanisms governing the catalytic activity of NANs. Additionally, it systematically reviews important research progress on NANs in colorimetric, fluorescent, electrochemical, SERS, and chemiluminescent sensors, offering insights into the development of the NAN field and biosensor applications.