This article describes the design and preparation of a novel fluorescence resonance energy transfer (FRET)-based ratiometric sensor with the polymer nanoparticle as scaffold for detecting Hg(2+) in aqueous media. In this study, a fluorescent dye fluorescein isothiocyanate (FITC, served as the donor) and a spirolactam rhodamine derivative (SRHB, served as mercury ion probe) were covalently attached onto polyethylenimine (PEI) and polyacrylic acid (PAA) respectively; and a ratiometric sensing system was then formed through the deposition of the donor- and probe-containing polyelectrolytes onto the negatively charged polymer particles via the layer-by-layer approach. The ratiometric fluorescent signal change of the system is based on the intra-particle fluorescence resonance energy transfer (FRET) process modulated by mercury ions. Under optimized structural and experimental conditions, the particle-based detection system exhibits stable response for Hg(2+) in aqueous media. More importantly, in this newly developed particle-based detection system formed by LBL approach, varied numbers of the PAA/PEI layers which served as the spacer could be placed between the donor-containing layer and the probe-containing layer, hence the donor-acceptor distance and energy transfer efficiency could be effectively tuned (from ca. 25% to 76%), this approach has well solved the problem for many particle-based FRET systems that the donor-acceptor distance cannot be precisely controlled. Also, it is found that the ratiometric sensor is applicable in a pH range of 4.6-7.3 in water with the detection limit of 200 nM. This approach may provide a new strategy for ratiometric detection of analytes in some environmental and biological applications.