A novel and sensitive biosensor based on aptamer and pyrene-labeled fluorescent probes for the determination of K+ was developed. The aptamer was used as a molecular recognition element and a partially complementary oligonucleotide with the aptamer was labeled by pyrene moieties at both ends to transduce the binding event of K+ with aptamer. In the presence of K+, the complementary oligonucleotides were displaced from aptamers, which was accompanied by excimer fluorescence of pyrenes because the self-hairpin structure of the complementary oligonucleotide brought pyrene moieties into close proximity. However, it gave only monomer emission in the absence of K+. Under optimum conditions, the relative fluorescence intensity of pyrene was proportional to the concentration of K+ in the range of 6.0 x 10(-4) to 2.0 x 10(-2) M. A detection limit of 4.0 x 10(-4) M was achieved. Moreover, this method was able to detect K+ with high selectivity in the presence of Na+, NH4+, Mg2+, and Ca2+ ions of biological fluids. In brief, the assay may have great potential applications, especially in a biological environment because of its simplicity, sensitivity, and specificity.