Mercury is a threatening pollutant in food, herein, we developed a Tb³⁺-nucleic acid probe-based label-free assay for mix-and-read, rapid detection of mercury pollution. The assay utilized the feature of light-up fluorescence of terbium ions (Tb³⁺) via binding with single-strand DNA. Mercury ion, Hg²⁺ induced thymine (T)-rich DNA strand to form a double-strand structure (T-Hg²⁺-T), thus leading to fluorescence reduction. Based on the principle, Hg²⁺ can be quantified based on the fluorescence of Tb³⁺, the limit of detection was 0.068 9 μmol/L and the linear range was 0.1‒6.0 μmol/L. Due to the specificity of T-Hg²⁺-T artificial base pair, the assay could distinguish Hg²⁺ from other metal ions. The recovery rate was ranged in 98.71%‒101.34% for detecting mercury pollution in three food samples. The assay is low-cost, separation-free and mix-to-read, thus was a competitive tool for detection of mercury pollution to ensure food safety.