A new ratiometric fluorescent nanoprobe based on DNA-stabilized silver nanoclusters (AgNCs) was one-pot synthesized by a simple reduction method. The obtained nanoprobe exhibited two typical green and red emissions of FAM and AgNCs, used as reference and response signals, respectively. Upon the addition of Hg²⁺ or Cu²⁺, the red emission of AgNCs was quenched greatly, whereas the green emission of FAM remained constant, resulting in the ratiometric fluorescence determination of Hg²⁺ and Cu²⁺. This ratiometric nanoprobe showed good selectivity for Hg²⁺ and Cu²⁺ over many other metal ions, and exhibited high sensitivity with a detection limit as low as 1.03 nM and 2.77 nM for Hg²⁺ and Cu²⁺, respectively. Moreover, with the aid of a chelating agent, EDTA, this ratiometric fluorescent nanoprobe can discriminate and realize exclusive determination of Hg²⁺ and Cu²⁺. This ratiometric fluorescent nanoprobe was successfully applied for the determination of Hg²⁺ and Cu²⁺ in real tap water samples.