The nanoimmunoassay chip comprises at least flow and control layers, divided into several rows, each row containing a plurality of single assay units, each assay unit contains two spotting chambers (1) and an assay chamber in the middle, wherein neck valves (2) separate the spotting chambers from the assay chamber during surface derivatization, said assay units being isolated from one another during incubation by isolation valves (3), wherein relief valves (4) help release built-up pressure into a microfluidic channel (5) after incubation and wherein round valves in the assay chamber define and protect the circular immunoassay regions (6).