We describe a chip-based immunoassay for multiplex antigen detection, based on the self-assembly of semi-synthetic DNA–protein conjugates to generate an easily configurable protein microarray. The general principle of this microarray-fluorescence immunoassay (μFIA) is similar to that of a two-sided (sandwich) immunoassay. However, covalent single-stranded DNA–streptavidin conjugates are employed for the efficient immobilization of biotinylated capture antibodies through hybridization to complementary surface-bound DNA oligomers. In a model system, we use the DNA-directed immobilization (DDI) of antibodies to generate an antibody microarray for the parallel detection of the tumor marker human carcinoembryonic antigen (CEA), recombinant mistletoe lectin rViscumin (rVis), ceruloplasmin (CEP), and complement-1-inactivator (C1 A) in human blood serum samples. Detection limits down to 400 pg mL−1are reached. In addition, we describe a method for the internal standardization of protein microarray analyses, based on the simultaneous measurement of constant amounts of the blood proteins CEP and C1 A, intrinsically present in human serum, to compensate for interexperimental variations usually occurring in microarray analyses. The standardization leads to a significantly higher data reliability and reproducibility in intra- and interassay measurements. We further demonstrate that the DDI-μFIA can also be carried out in a single step by tagging of the analyte simultaneously with both capture and detection antibody and subsequent immobilization of the immunocomplex formed, on the DNA microarray capture matrix. This protocol significantly reduces handling time and costs of analysis.