Nucleic acids have been actively exploited to develop various exquisite nanostructures due to their unparalleled programmability. Especially, framework nucleic acids (FNAs) with tailorable functionality and precise addressability hold great promise for biomedical applications. In this review, we summarize recent progress of FNA-enabled biosensing in homogeneous solutions, on heterogeneous surfaces, and inside cells. We describe the strategies to translate the structural order and rigidity of FNAs to interfacial engineering with high controllability, and approaches to realize multiplexing for highly parallel in vitro detection. We also envision the marriage of the currently available FNA tool sets with other emerging technologies to develop a new generation of biosensors for precision diagnosis and bioimaging.