Reduction of hydrophobic interaction in water is important in biological interfaces. In our previous work, we have found that poly (styrene-b-triethylene glycol methyl ether methacrylate) (PS-PME3MA) segregates the PME3MA block to the surface in hydrophobic environment, such as in air or in a vacuum, and shows remarkable resistance against adsorption or adhesion of proteins, platelets, and cells in water. In this paper, we report that atomic force microscopy (AFM) with hydrophobic probes can directly monitor the reduced hydrophobic interaction of the PS surfaces modified by poly(styrene-b-origoethylene glycol methyl ether methacrylate) (PS-PMENMA), where N is the number of ethylene glycol units. The pull-off forces between the hydrophobic probes that are coated with octyltrichlorosilane (OLTS) and the PS-PMENMA modified polystyrene (PS) surfaces in water were measured. The absolute spring constants and tip-curvatures of the AFM cantilevers were measured to compute the work of adhesion by the Johnson, Kendall, and Roberts (JKR) theory, which relates the pull-off force at which the separation occurs between a hemisphere and a plane to the work of adhesion. The hydrophobic interactions between the hydrophobic tip and polymer surfaces in water were greatly reduced with the segregated PMENMA blocks. The hydrophobic interactions decrease with increasing N of the series of PS-PMENMA and show a correlation with the amount of protein adsorbed.