A wide range of cadmium sulfide (CdS) 3D polycrystalline walnut-like nanocrystals were prepared by solvothermal method with polyvinylpyrrolidone (PVP) as stabilizer. The morphology, structure, and phase composition of the as-prepared CdS nanoparticles were examined by using various techniques (X-ray diffraction (XRD), field-emission scanning electron microscopy (FESEM), transmission electron microscopy (TEM), selected-area electron diffraction (SAED)). The optical properties of CdS were characterized by photoluminescence (PL) spectra. The paper showed that when the proper dosage of PVP (0.12 g 50 mL −1) and the temperature of reaction (120 °C) were taken, continuous polycrystalline nanoparticles with regular morphology were obtained. The mechanism for the PVP-assisted synthesis of CdS 3D nanostructures and the growth process of CdS nanoparticles via Ostwald Ripening process under different temperatures with different dosages of PVP were also investigated. A sensitive electrochemical detection device of DNA hybridization using a paste glass carbon electrode (GCE) assembled by the PVP-capped CdS nanoparticles and immobilizing DNA probe was developed as a novel application of nano-semiconductors such as CdS to achieve a notable amplification in sensor response.