A novel nanoparticles-based fluorescence detection method has been developed by taking advantage of magnetic separation and amplified fluorescence detection. This DNA sensor relies on a "sandwich" hybridization strategy, in which the DNA targets are first hybridized to captured oligonucleotide probes immobilized on magnetic nanoparticles, and then hybridized with thiol-modified oligonucleotide probes immobilized on gold nanoparticles. Subsequently, the amplified DNA signals are detected in the form of bio-bar-code DNA using a chip-based fluorescence detection method. The result showed that the detection limit of target DNA probes is 1 pM. Complementary and mismatched sequences were clearly distinguished, and the ratio of the background-subtracted fluorescence values for complementary and single-base mismatched oligonucleotide was 2.12:1. This new system can be applied to both DNA detection and immunoassay, and has broad potential applications in disease diagnosis and immunoassay.