Dual-emission nanosensor for Hg2+ detection was prepared by coupling CA-AEAPMS on the surface of RBS-doped modified silica microspheres. The CA-AEAPMS was synthesized by using N-(β-aminoethyl)-γ-aminopropyl methyldimethoxysilane (AEAPMS) and citric acid as the main raw material. The obtained nanosensor showed characteristic fluorescence emissions of Rhodamine B (red) and CA-AEAPMS (blue) under a single excitation wavelength (360 nm). Upon binding to Hg2+, only the fluorescence of CA-AEAPMS was quenched, resulting in the ratiometric fluorescence response of the dual-emission silica microspheres. This ratiometric nanosensor exhibited good selectivity to Hg2+ over other metal ions, because of the amide groups on the surface of CA-AEAPMS serving as the Hg2+ recognition sites. The ratio of F450/F580 linearly decreased with the increasing of Hg2+ concentration in the range of 0 to 3 × 10−6 M, and a detection limit was as low as 97 nM was achieved. Then, the addition of three thiol-containing amino acids (Cys, Hcy, GSH) to the quenched fluorescence solution with Hg2+ can restore the fluorescence, and the detection limits of the three biothiols (Cys, Hcy, GSH) are 0.133 μM, 0.086 μM, and 0.123 μM, respectively.