Antibody microarrays have shown great potential for measurement of either a spectrum of target proteins in proteomics or disease-associated antigens in molecular diagnostics. Despite its importance, the applications of antibody microarrays are still limited by a variety of fundamental problems. Among them, cross-reactivity significantly limits the multiplexing ability in parallel sandwich immunoassays. As a result, it is very important to design new capture probes in order to incorporate a universal label into the assay configuration. In this report, an antibody fragments (F(ab′)₂) microarray platform for serum tumor markers was developed. Each antigen was detected at different concentrations to assemble its calibration curve, and combinations of different markers were tested to examine the specificity of simultaneous detection based on the F(ab′)₂ microarrays. Diagnostics of serum samples with this cancer antibody microarray platform and immunoradiometric assays (IRMA) were also performed. Wide range calibration curves (0–1280 U mL^–1) were obtained for each tumor marker. Comparative studies demonstrated that such F(ab′)₂ microarrays exhibited both moderately improved sensitivity and better specificity than full-sized monoclonal antibody microarrays. It is also demonstrated that this microarray platform is quantitative, highly specific and reasonably sensitive. More importantly, clinical applications of our F(ab′)₂ microarray platform for upwards of 100 patient serum samples clearly show its potential in cancer diagnostics.