We have developed a fast and sensitive immunoassay for the determination of bisphenol A (BPA), using the fluorescence quenching effect between gold nanoparticles and fluorescein isothiocyanate (FITC). Herein, carboxyl-modified single-strand DNA (COOH-ssDNAs) and anti-BPA antibodies were simultaneously conjugated with silica nanoparticles, and FITC-labeled single-strand DNA (FITC-ssDNA, the complement of COOH-ssDNA) was hybridized with COOH-ssDNA to form a signal platform, and the BPA coated antigen-functionalized nanogold was regarded as an acceptor. In the presence of BPA, a competitive immunoreaction takes place between BPA and BPA coated antigen-functionalized nanogold for the binding sites of the anti-BPA antibodies on the signal platform. Due to the fact that the photoluminescence of FITC was strongly quenched by the AuNPs, the fluorescence emission was decreased significantly. Under optimized conditions, the fluorescence intensity had a linear signal response range with a BPA concentration from 2.0 × 10⁻³ ng mL⁻¹ to 1.0 ng mL⁻¹ with a low limit of detection of 1.44 × 10⁻³ ng mL⁻¹. Furthermore, this new signal platform was successfully applied to detect real samples for low levels of BPA.