A sensitive chemiluminescence (CL) imaging immunoassay method for detection of multiple tumor markers with high throughput, easy operation, and low cost was developed. The immunosensor array was prepared by covalently immobilizing capture antibodies on corresponding sensing sites on a silanized disposable glass chip. Gold nanoparticle-based bioconjugates with a high molar ratio of horseradish peroxidase (HRP) to detection antibodies were used for signal amplification. Under a sandwich immunoassay, the CL signals triggered by HRP captured on each sensing cell were collected by a charge-coupled device for simultaneous measurement of biomarkers and combination diagnosis of certain tumors. As a proof of concept, the immunosensor array was applied to detect α-fetoprotein, carcinoma antigen 125, carbohydrate antigen 153, and carcinoembryonic antigen and to screen patients with liver, breast, or ovarian cancers. This method showed wide linear ranges over 5 orders of magnitude and much lower detection limits than previously reported multiplexed immunoassays. The high throughput and acceptable stability, reproducibility, and accuracy showed good applicability of the proposed multiplex CL imaging immunoassay in clinical diagnosis.