Abstract: A series of comb-like poly(2-methyl-2-oxazoline)-random-glycidyl methacrylate (PMeOx-r-GMA) with PMeOx side chains of molecular weights of approximately 1.0×10³, 2.0×10³, and 6.0×10³ were synthesized by using cationic ring-opening polymerization and free radical copolymerization. Then, the double-brush coatings based on PMeOx on silicon/glass/polyurethane surfaces were constructed by heating the mixture of PMeOx-r-GMA with two different PMeOx chain lengths. The coatings were characterized by water contact angle (WCA), variable-angle spectroscopic ellipsometry (VASE), and atomic force microscopy (AFM). Fluorescence microscopy and scanning electron microscopy (SEM) were used to investigate the adsorption of proteins, platelets, and bacteria on the coating surfaces. Protein adsorption results showed that the amount of protein adsorption on the double-brush coatings decreased compared to the coatings made of PMeOx with a single chain length. The amount of larger proteins like human serum immunoglobulin/bovine serum albumin adsorbed on double-brush coatings reduced more with a large difference in PMeOx chain length, while the amount of smaller protein adsorption like lysozyme reduced more on double-brush coatings with a smaller difference in PMeOx chain length. In addition, the amount of platelets adsorption is minimum on double-brush coatings with a large difference in PMeOx chain length, while E. coli and S. aureus adsorbed is minimum on double-brush coatings with a small difference in PMeOx chain length. In addition, after soaking the double-brush coatings formed on glass/polyurethane surfaces in phosphate-buffered solution for 14 days, relative fluorescence intensity of adsorbed bovine serum albumin only increased by at most 1.50% and 0.76%, respectively, indicating that the coatings have long-term stability.