Sensitive DNA detection is extremely important in clinical diagnostics, gene therapy, and a variety of biomedical studies. We have developed a novel DNA bioanalysis method with a 0.8 fM (0.8 x 10-15 M) detection limit using a bioconjugated fluorescent nanoparticle-based sandwich assay. An organic dye silica nanoparticle is synthesized using a modified reverse microemulsion. The nanoparticles are highly fluorescent, extremely photostable, and easy for bioconjugation for bioanalysis. They exhibit an excellent signaling ability in the presence of trace amounts of DNA targets. With an effective surface modification, nonspecific binding and nanoparticle aggregation are minimized. In addition, the nanoparticle-based DNA bioanalysis assay can effectively discriminate one-base mismatched DNA sequences. We expect this nanoparticle-based assay to be widely useful in a number of biomedical applications where reproducible, selective, and ultrasensitive gene analysis is critical.