When the materials get contact with blood, non-specific protein adsorption occurs on the surface and the protein adsorption layer induces much severe biological responses including cellular reactions. Therefore, evaluating the first protein adsorption is important for preparing the nonbiofouling surfaces. Atomic force microscopy (AFM) makes it possible to directly measure the forces generated from protein-surface interaction in aqueous media down to few piconewtons range. In this study, we evaluated the adsorption force between bovine serum albumin (BSA)-immobilized AFM cantilever and well-defined polymer brush surface with three kinds of hydrophilic monomers, which are 2-methacryloyloxyethyl phosphorylcholine (MPC), poly (ethylene glycol) methacrylate (PEGMA) and 2-hydroxyethyl methacrylate (HEMA) by surface-initiated atom transfer radical polymerization (SI-ATRP) on initiator-immobilized silicon wafer in buffer solution. We found that the protein adsorption forces depended on the both chemical structure and thickness of the polymer brush layer. In particularly, the thickness was important to reduce protein adsorption force. From these results, thick polymer brush surface is one of the candidate surfaces with nonbiofouling characteristics.