This paper describes a simple method for preparing protein microarrays that is compatible with high throughput manufacturing, The microarrays were formed by maskless photolithography and pin spotting to study protein adsorption on a fluorinated/methoxy-poly(ethylene glycol) (PEG) self-assembled monolayer (SAM). The mixed nonionic surfactants, Tween20 and PEG200, were utilized to control nonspecific protein adsorption oil both of the SAMs. Measurements using double-antibody sandwich quantum dots-linked immunosorbent assay (DAS-QDLISA) showed that the fluorinated SAM could effectively minimize nonspecific adsorption in the presence of Tween 20 and PEG 200 (inhibitors of nonspecific protein adsorption (INSPAs)) while the PEGylated surface was biofouling. Additionally, pin spotting was used to fabricate high-throughput protein arrays oil the fluorinated SAM. The results displayed that fluorinated SAM could not only effectively immobilize protein by hydrophobic interactions, but could also resist the other nonspecific adsorption with the INSPA. In this way, protein microarrays would be formed more conveniently and environmentally friendly. It is believed that simple, practical, and high-throughput protein immunosensing could be established with these mixed nonionic surfactants.