Nucleic acids can be used as building blocks for highly regular and complex structures at the nanoscale due to their inherent self-assembly and programmability resulting from intra- and inter-molecular interactions. Three-way junctions (TWJs) structure is recognized as the smallest type of junction in nucleic acids. To form TWJ nanostructures, three DNA or RNA strands with complementary segments at each end are hybridized and form a Y-shaped structure. TWJ skeletons, having three independent arms, provide multifunctional sites for beneficial amplification, recognition, and signaling purposes in specific biomolecular nanoarchitecture. In this study, we represent the superiority aspects on the achievements of the biosensors with the cooperation of DNA/RNA TWJ nanoskeletons for monitoring different targets. Moreover, some discussions are declared to achieve a perspective for developing promising TWJ-based aptasensors in the future. This in-depth study can open a constructive way for commercializing and miniaturizing TWJ-tuned aptasensors as user-friendly lab-on-chip sensing devices. Display omitted •Nano-biotechnology has emerged as a promising route in biosensing engineering by representing the diverse DNA/RNA skeletons as smart recognition elements.•DNA/RNA skeletons based on three-way junction (TWJ) structures provide multifunctional sites for beneficial amplification, recognition, and signaling purposes.•Biosensors based on TWJ structures are ease-of-use, real-time, cost-effective, and label-free non-enzymatic sensing tools for the diagnosis of diverse targets.•We represent the superiority aspects on the achievements of biosensors with the cooperation of TWJ nano-skeletons.