Enzyme-linked immunosorbent assays (ELISA) show extensive application in immunoassays, to detect and monitor protein biomarkers in clinical diagnosis. Nevertheless, the time required and its multiple steps limit its application. We take advantage of a polyethyleneimine (PEI) gold nanoparticle (GNP) coated microwell plate to perform blocking-free ELISA, in which no nonspecific protein adsorption appears on the GNP layer. If the PEI-GNP coated microwell plate and immobilization of captured antibodies on the plate are prepared in advance, such as using an ELISA kit, the whole ELISA process can be finished in less than 2 h. Meanwhile, we have ensured that the GNP layer can preserve the precision and good linearity of ELISA without causing negative effects on the plate.