Abstract: A fluorescent aptamer probe was developed for highly sensitive sensing detection of microcystin-LR (MC-LR) based on poly(adenine) DNA-gold nanoclusters (poly(A)-AuNCs) using DNA template method. Three DNA nucleotides were designed in this work, including MC-LR aptamer, poly(adenine) DNA S1 (poly(A) S1), and complementary DNA S2 of aptamer. Using poly(A) S1 as a template, HAuCl₄ was reduced with sodium citrate to synthesize poly(A)-AuNCs with blue fluorescence. MC-LR aptamer hybridized with poly(A)-AuNCs and DNA S2 to form dsDNA-AuNCs. Photo-induced electron transfer (PET) occurred between AuNCs (electron donors) and G-bases (electron acceptors) of DNA S2 in the dsDNA-AuNCs structure, resulting in fluorescence quenching of dsDNA-AuNCs. In the presence of target MC-LR, it specifically binds to aptamer in dsDNA-AuNCs, leading to dissociation of dsDNA structure. Poly(A)-AuNCs were released into the solution, restoring the intensity of fluorescence. An "off-on" fluorescence system was constructed for MC-LR detection. The linear range of the fluorescence probe for MC-LR detection is 5 ng/L ~ 100 μg/L, with a detection limit of 1.5 ng/L (S/N = 3). This fluorescent aptamer probe has the advantages of simple preparation, high selectivity and sensitivity, and can be applied for quantitative analysis of MC-LR in actual water samples.