A new method has been developed for the simple, fluorescence, turn-on detection of melamine, which utilizes DNA-templated silver nanoclusters (DNA-AgNCs) as a key component. In the sensor, melamine exhibits the dual functions: one is to enhance the fluorescence signal of DNA-AgNCs by its specific interaction with thymine residues in DNA template, and the other is to prevent Hg(II)-induced fluorescence quenching of DNA-AgNCs via its strong coordination with Hg(II). These consequently enable the sensitive and selective detection of melamine. By exploiting such novel features of melamine, we significantly increased the fluorescence response up to 360%, compared to the previous counterpart that relies on DNA-AgNCs only, and successfully determined melamine down to ca. 49 nM, a value that is 400 times lower than the safety level of 20 μM set by the US Food and Drug Administration. In addition, it was confirmed that the proposed approach works fine even in the real milk samples without any additional pre-treatment steps.