Research on self-assembled deoxyribonucleic acid (DNA) nanostructures with different shapes, sizes, and functions has recently made rapid progress owing to its biocompatibility, programmability, and stability. Among these, triangular unit-based DNA nanostructures, which are typically multi-arm DNA tiles, have been widely applied because of their unique structural rigidity, spatial flexibility, and cell permeability. Triangular unit-based DNA nanostructures are folded from multiple single-stranded DNA using the principle of complementary base pairing. Its shape and size can be determined using pre-set scaffold strands, segmented base complementary regions, and sequence lengths. The resulting DNA nanostructures retain the desired sequence length to serve as binding sites for other molecules and obtain satisfactory results in molecular recognition, spatial orientation, and target acquisition. Therefore, extensive research on triangular unit-based DNA nanostructures has shown that they can be used as powerful tools in the biosensing field to improve specificity, sensitivity, and accuracy. Over the past few decades, various design strategies and assembly techniques have been established to improve the stability, complexity, functionality, and practical applications of triangular unit-based DNA nanostructures in biosensing. In this review, we introduce the structural design strategies and principles of typical triangular unit-based DNA nanostructures, including triangular, tetrahedral, star, and net-shaped DNA. We then summarize the functional properties of triangular unit-based DNA nanostructures and their applications in biosensing. Finally, we critically discuss the existing challenges and future trends.