The instant and on-site detection of selenium still remains a challenge for environmental monitoring and medical prevention. We herein developed a ratiometric fluorescent nanosensor for accurate and on-site sensing of SeO₃²⁻ by linking the recognition molecule 3,3′-diaminobenzidine (DAB) onto the surface of carboxyl group modified CdTe@SiO₂. The fluorescence of DAB on the surface of silica nanospheres could be selectively and efficiently enhanced by SeO₃²⁻ through a surface chelating reaction between DAB and SeO₃²⁻. Thus, in the presence of SeO₃²⁻, the nanosensor would show two characteristic fluorescence emissions of Se-DAB and CdTe QDs under a single excitation wavelength. The selectivity and the optimal conditions for the detection of SeO₃²⁻ were carefully investigated. The ratio of F₅₃₀/F₆₃₅ linearly increased with increasing SeO₃²⁻ concentration in the range of 0 to 2.5 μM and the detection limit reaches as low as 6.68 nM (0.53 ppb). This developed nanosensor has the capability of on-site detection in an aqueous system without any separation step. The Se concentrations in selenium-rich food were detected and the results were consistent with the values determined by ICP-AES.