DNA-immobilized nanoparticle probes show high target specificity; thus, they are employed in various bioengineering and biomedicine applications. When the nanoparticles employed are dye-loaded polymer particles, the resulting probes have the additional benefit of biocompatibility and versatile surface properties. In this study, we construct DNA-immobilized fluorescent polystyrene (PS) nanoparticles through controlled surface reactions. PS nanoparticles with surface carboxyl groups are utilized, and amine-functionalized dye molecules and capture DNAs are covalently immobilized via a one-pot reaction. We show that the surface chemistry employed allows for quantitative control over the number of fluorescent dyes and DNA strands immobilized on the PS nanoparticle surfaces. The nanoparticles thus prepared are then used for DNA detection. The off state of the nanoprobe is achieved by hybridizing quencher-functionalized DNAs (Q-DNAs) to the capture DNAs immobilized on nanoparticle surfaces. Target-DNAs (T-DNAs) are detected by the displacement of the prehybridized Q-DNAs. The nanoprobes show successful detection of T-DNAs with high sequence specificity and long-term stability. They also show excellent detection sensitivity, and the detection limit can be tuned by adjusting the capture DNA-to-dye ratio.