A novel and label-free method for micrococcal nuclease (MNase) detection has been presented based on single-stranded DNA (ssDNA)-scaffolded fluorescent silver nanoclusters (AgNCs). The ssDNA was introduced as the substrate for MNase and also as the scaffold for the synthesis of the AgNCs. In the absence of MNase, the ssDNA was not digested. As a result, the fluorescent AgNCs were formed and exhibited strong fluorescence. In the presence of MNase, the DNA was digested, which prohibited the formation of the AgNCs due to the lack of the DNA scaffold, resulting in weak fluorescence. The fluorescence intensity exhibits a linear correlation to MNase concentration in the range of 0 U mL⁻¹ to 2 × 10⁻⁴ U mL⁻¹ with a detection limit of 8 × 10⁻⁶ U mL⁻¹. Given its simplicity, easy operation, sensitivity and cost-effectiveness, this method can be extended to other nuclease assays.