We herein report an ultralow background substrate for protein microarrays. Conventional protein microarray substrates often suffer from non-specific protein adsorption and inhomogeneous spot morphology. Consequently, surface treatment and a suitable printing solution are required to improve the microarray performance. In the current work, we improved the situation by developing a new microarray substrate based on a fluorinated ethylene propylene (FEP) membrane. A polydopamine microspot array was fabricated on the FEP membrane, with proteins conjugated to the FEP surface through polydopamine. Uniform microspots were obtained on FEP without the application of a special printing solution. The modified FEP membrane demonstrated ultralow background signal and was applied in protein and peptide microarray analysis.