Recent advancements in DNA nanotechnology have unlocked unprecedented opportunities to address critical challenges in precision medicine, particularly in targeted drug delivery and biomedical imaging. Conventional nanocarriers often suffer from poor spatiotemporal control, suboptimal tumor accumulation, and non-specific biodistribution. To overcome these limitations, DNA-engineered nanostructures-including tile-based assemblies, origami frameworks, spherical nucleic acids, and stimuli-responsive hydrogels-have emerged as programmable platforms capable of dynamically responding to tumor microenvironmental cues (, pH, enzymatic activity, redox gradients) for triggered drug release. In this review, we comprehensively analyze these architectures with emphasis on their modular design strategies, stability improvements polyethylene glycol (PEG) functionalization, and multi-ligand targeting capabilities against cancer-specific biomarkers. In addition to therapeutic uses, these nanostructures also enable highly sensitive detection of circulating tumor DNA and exosomes using fluorescence resonance energy transfer (FRET) probes, electrochemiluminescence amplification circuits, SERS substrates, and cell variable region sensing technology. They also allow for real-time monitoring of dynamic intercellular interactions, overcoming the constraints of traditional sensing methods. This review systematically elaborates on the structural characteristics of DNA assemblies and summarizes the innovative applications of these nanostructures in multimodal detection, offering a more comprehensive perspective for early cancer diagnosis and precision treatment. Despite promising preclinical results, key translational challenges persist, including scalable manufacturing bottlenecks, immune compatibility optimization, and rigorous assessment of long-term nanotoxicity. Future integration with artificial intelligence-driven design tools may catalyze the development of next-generation theranostic nanodevices, ultimately bridging the gap between synthetic biology and clinical oncology.