A homogenous fluorescence dual-probe assay containing 2′-N-(pyren-1-ylmethyl)-2′-amino-LNA (locked nucleic acid) building blocks has been developed for effective mismatch-sensitive nucleic acid detection. The pyrene units, which are connected to the rigid bicyclic furanose derivative of 2′-amino-LNA through a short linker, are positioned at the 3′ and 5′ ends of a dual-probe system. Whereas hybridization with complementary DNA/RNA results in very strong excimer signals, as the pyrene units are in close proximity to one another in the ternary complex, exposure to most singly mismatched DNA/RNA targets results in significantly lower excimer emission intensity. The mechanism that underlies this excellent optical discrimination of singly mismatched targets is clarified by comparison of the thermal-denaturation profiles and fluorescence properties of the dual probe and a covalently linked analogue. Optical discrimination of singly mismatched targets arises from a decrease in excimer emission intensity due to a failure to form a ternary complex (a decrease in thermal stability) and/or local mismatch-induced changes in the helix geometry, depending on the position of the mismatched base pair. The devised dual-probe assay constitutes a simple and sensitive system for the detection of single-nucleotide polymorphism and highlights that conformational restriction combined with the use of short probes conveys favorable properties to dual-probe constructs.