<p>Human telomeric DNA sequences (5′-TTAGGGTTAGGGTTAGGGTTAGGG-3′) can be induced by strontium ions to form G-quadruplex structures. Based on this property, a novel “OFF-ON” fluorescent sensor was developed for Sr²⁺ using a G-quadruplex/acridine orange-graphene oxide (G4/AO-GO) system. In the presence of Sr²⁺, the telomeric DNA folds into a rigid G-quadruplex structure, which not only inhibited the adsorption of acridine orange (AO) on graphene oxide (GO) and recovered the fluorescence of AO, but enhanced the fluorescence of AO as well. Spectroscopic results confirmed the formation of stable G-quadruplexes in the presence of Sr²⁺. Under the optimum condition, the fluorescence intensity showed a good linear relationship with the Sr²⁺ concentration over the range from 10 nmol/L to 3.0 µmol/L, and the detection limit was 3.6 nmol/L. The sensor also exhibited high specificity and selectivity, and was successfully applied to the determination of strontium in mineral water samples. This sensing platform provides a promising method for <i>in situ</i> monitoring of Sr²⁺ in geological surveys, environmental safety assessment, and food quality control.</p>