Abstract: Double stranded DNA-copper nanoclusters (dsDNA-CuNCs) fluorescent aptamer probe was prepared for highly sensitive sensing detection of microcystin-LR using DNA-template method. Microcystin-LR aptamer hybridized with its complementary strand cDNA to form double stranded DNA (dsDNA). Using dsDNA as a template, copper ions (Cu²⁺) were reduced by ascorbic acid to form double stranded DNA-copper nanoclusters (dsDNA-CuNCs) probe with pink fluorescence. In the presence of microcystin-LR (MC-LR) object, fluorescence of dsDNA-CuNCs probe was quenched due to the specific binding between MC-LR and aptamer in dsDNA-CuNCs, resulting in dissociation of dsDNA and releasing of CuNCs into the solution. And the cyanine-5 fluorophore labeled cDNA (Cy5-cDNA) interacted with the released CuNCs, leading to the fluorescence quenching of Cy5 cDNA simultaneously. Therefore, a dual-mode fluorescence quenching system was constructed. The proposed method exhibited a linear range of 10 ng/L to 500 μg/L for MC-LR detection with a detection limit of 3.3 ng/L (S/N = 3). This fluorescent aptamer probe has the advantages of simple preparation and dual-mode detection, which can be applied for the detection and analysis of MC-LR in actual water samples.