Developing new technologies applicable to the sensitive detection of cancer in its early stages has always been attractive in diagnosis. A stable gold nanoparticle layer (GNPL)-modified high-binding ELISA plate was obtained via chemical plating and was proven to be more efficient in binding proteins while maintaining their activity. GNPL-based ELISA for the representative biomarker carcinoembryonic antigen (CEA) demonstrated that GNPL markedly amplified the ELISA signal and significantly improved the limit of detection (LOD). Antithrombin detection further confirms the effectiveness and universality of this GNPL-based platform. The entire assay procedure is simple and low in cost and does not require special facilities. All these virtues indicate that this GNPL platform holds great promise in clinical applications for the early diagnosis of cancer.