Antibody-based cell microarrays may serve as a high-throughput diagnostic tool that requires precise surface design providing high biological specificity, high reliability, and high validity. A fundamental study on the quantitative evaluation of immunophenotyping using a cell microarray on which antibodies specific to cluster of differentiation (CD) antigens of blood cells are fixed was performed. The microarray, which was prepared by photomicropatterning self-assembled monolayers of alkanethiols, consisted of carboxyl group (3-mercaptopropionic acid)-packed domains regularly distributed in the methyl group (1-dodecanethiol)-packed matrix phase. This was verified by X-ray photoelectron spectroscopy and water wettability measurements. The patterned carboxylated domains of 1.0 mm or 0.2 mm diameter were covalently fixed with the anti-CD4 or anti-CD8 antibody by coupling reaction using a water-soluble carbodiimide and hydroxysuccinimide. Precision antibody fixation at almost complete conversion was verified by confocal laser scanning microscopy coupled with a specific dye staining technique. Peripheral blood mononuclear cells expressing CD4 or CD8 antigen, respectively, adhered on the anti-CD4 or anti-CD8 antibody-fixed domains of the microarray at a very high specificity, which was verified with flow cytometric analysis and an antibody-coated magnetic-bead-based cell isolation technique. The CD4/CD8 subset ratios determined using the antibody-fixed microarrays were very close to those obtained by flow cytometric analysis. These results indicate that microarrays, on which antibodies specific to cell surface antigens are covalently fixed, provide a solid basis of the high-throughput quantitative evaluation of immunophenotyping in medical diagnosis.