The development of antifouling surfaces is essential for many biomedical applications such as diagnostic assay materials, biosensors, blood contacting devices, and other implants. In this study, a facile one-step surface modification strategy to modify inorganic and organic substrate surfaces by dopamine-assisted immobilization of partially hydrolyzed poly(2-methyl-2-oxazoline) [P(MOXA-co-EI)] was established. Firstly, poly(2-methyl-2-oxazoline)s (PMOXAs) with different molecular weights were synthesized by cationic ring-opening polymerization, and acidic hydrolysis of PMOXA was performed to get the P(MOXA-co-EI)s with different hydrolysis degrees, and then the mixed solutions of P(MOXA-co-EI) and dopamine (DA) with different mass concentration ratios of P(MOXA-co-EI) to DA, different molecular weights and hydrolysis degrees of PMOXA were used as co-deposited coatings to modify glass/silicon/poly(dimethylsiloxane) (PDMS) surfaces. The obtained co-deposited coatings were rigorously characterized in terms of the surface chemical composition, thickness, morphology, hydrophilicity, and stability by X-ray photoelectron spectroscopy, ellipsometry, atomic force microscopy, and the contact angle measurements. The results demonstrated that the coatings could be successfully deposited onto silicon/glass/PDMS surfaces and possessed good stability. Finally, the fouling resistance ability and blood compatibility of the modified substrates were evaluated by analyzing the adsorption of bovine serum albumin protein (BSA) and platelet adhesion, respectively. The results showed that the co-deposited coating-modified surfaces had a superior resistance to the adsorption of BSA and platelet when the molecular weight of PMOXA was 0.8 or 2 kDa, the hydrolysis degree was 0.12, and the concentration ratio of P(MOXA-co-EI) to DA was enhanced to 2/1. The human umbilical vein endothelial cells attachment test suggested that the co-deposited coating could enhance the cell adhesion and growth on modified substrates, endowing biomaterials with well cytocompatibility.