In this study, a novel dual-emission ratiometric fluorescent nanoprobe (RFN) was synthesized and ultilized for highly sensitive determination of mercury ions. In this nanoprobe, fluorescein isothiocyanate (FITC) doped silica (SiO2) served as a reference signal, FITC–SiO2 microspheres were synthesized and modified with amino groups, and then Au Nanoclusters (AuNCs) were combined with the amino groups on the surface of the FITC–SiO2 microspheres to obtain the RFN. The selectivity, stability, and pH of the RFN were then optimized, and the determination of mercury ions was performed under optimal conditions. The probe fluorescence intensity ratio (F520 nm/F680 nm) and Hg2+ concentration (1.0 × 10−10 mol/L to 1.0 × 10−8 mol/L) showed a good linear relationship, with a correlation coefficient of R2 = 0.98802 and a detection limit of 1.0 × 10−10 mol/L, respectively. The probe was used for the determination of trace mercury ion in water samples, and the recovery rate was 98.15~100.45%, suggesting a wide range of applications in monitoring pollutants, such as heavy metal ion and in the area of environmental protection.