Abstract: An enzyme-free and label-free fluorescence sensing method for microcystin-LR detection was developed based on single-stranded DNA-silver nanoclusters (ssDNA-Ag NCs) fluorescent probe, which was prepared by using ssDNA as template. The designed ssDNA can not only be used as a template to synthesize ssDNA-Ag NCs fluorescent probe, but also bind to microcystin-LR through high affinity and high specificity. The morphology and optical properties of prepared ssDNA-Ag NCs were characterized by transmission electron microscope (TEM), ultraviolet–visible spectroscopy (UV-vis) and fluorescent spectroscopy (FL). The experimental results showed that fluorescence intensity of ssDNA-Ag NCs quenched in the presence of microcystin-LR because the affinity between ssDNA and microcystin-LR was stronger than that of Ag NCs resulted in the release of Ag NCs from ssDNA. Under the optimized experimental conditions, the linear range of ssDNA-Ag NCs fluorescent probe for microcystin-LR detection was 0.05−1000 µg/L with a detection limit of 16 ng/L (S/N = 3). The developed fluorescent probe has the advantages of simple preparation, no labeling and high sensitivity, and provides a simple, reliable and effective method for the determination of microcystin-LR in practical environmental water samples.