The measurement of biomarkers in bodily fluids is extremely important for diagnosing disease, monitoring disease progression, and evaluating treatment efficacy. In this paper, we present a highly sensitive and compatible gold nanoparticle (AuNP)-based, two-step signal amplification system for biomarker detection. First, AuNPs were coated onto the surfaces of 96-well plates to generate rough surfaces, which enable immobilization of many more capture antibodies than a smooth substrate. As a result, detection sensitivity was enhanced significantly. Second, the horseradish peroxidase (HRP)-conjugated detection antibodies were labeled on large-size AuNPs, which increase the localized amounts of HRP and thus further lower the detection limit. Based on the consecutive signal amplification system, a high-sensitivity assay was achieved, with a LOD of 0.07 ng/mL for prostate-specific antigen (PSA). This assay was allowed to detect the PSA levels in clinical samples without changing the current standard immunoassay setups, showing great potential in many settings where immunoassays are needed.