Enzyme-linked immunosorbent assays are routinely used in laboratories around the world and ensure highly specific protein detection. Often, more than one analyte needs to be determined in a single sample and numerous protein arrays for multianalyte detection of a single sample have been developed to address this problem. They have the potential to analyze several dozen or even more analytes in an assay volume of usually around 100 μL. However, due to the presence of numerous different antibodies, these multianalyte sandwich immunoassays suffer from undesired cross-reactivities between the antibodies which lead to a loss of assay specificities. Here, we present an assay principle which allows, e.g., a detection of an analyte in a sample volume of only 1 μL in a normal 96-microtiter well plate, so that up to 100 analytes can be determined from a 100 μL sample volume, but in separate wells. This eliminates antibody cross-reactivities. The assay is based on the biotinylated time-resolved fluorophore EuLH used as a PEG11-dye conjugate in combination with ExtrAvidin® to ensure high signal-to-background ratios. The model protein epidermal growth factor (EGF) was detected with the established sandwich immunoassay and showed assay parameters comparable to commercially available ones. Furthermore, the assay principle enables a spatial resolution of the assay signal. Here, we demonstrated the application of the new detection system for universal imaging-based analysis of individual spots in one single 96-microtiter well by applying it to multisample and also multianalyte detections. In the case of the multisample analysis approach, a considerable reduction of the required sample volume to only 1 μL in a single 96 microtiter well could be achieved.