The highly specific and highly sensitive ELISA (enzyme linked immunosorbent assay) technique is the most commonly used method for immunological diagnostics in general. In combination with protein microarrays and their ability to allow performing thousands of experiments in parallel, a promising tool for global analytical approaches with reduced consumption of time, analytes, and reagents is given. In this study a protein microarray-based sandwich-ELISA for human interferon-gamma (hINF-gamma) is established. In consideration of the immense importance of the surface chemistry, a new black nitrocellulose matrix that generates very high signal-to-noise ratios (SNR) and a very low autofluorescence was tested and optimized as microarray substrate. A validation of the applicability of the system was performed with a comparison to different commercially available systems. Experimental results show that the microarray-based ELISA is faster and easier to perform and shows a lower limit of detection (LOD) than a comparable system in a 96-well plate. The spotted slides with the capture antibody can be stored up to 1 month with no significant loss of signal intensity. A second model system with immobilized His-tagged restriction enzyme EcoRV and an anti-His antibody shows in coincidence the good applicability of the black nitrocellulose membrane and no cross-reactivity toward the ELISA.