Immobilization is widely used to isolate agglutinative and associative proteins with large hydrophobic surfaces. Surface hydrophobicities of immobilized proteins were quantified by measuring the adsorption amounts of Triton X-100 as a hydrophobic probe with a biosensor that utilizes the phenomena of surface plasmon resonance (SPR). We measured SPR signal changes derived from adsorption of Triton X-100 to five kinds proteins and calculated the monolayer adsorption capacity using the Brunauer–Emmett–Teller equation, partly modified with a term for correcting an influence of the net charge of immobilized protein. SPR signal changes obtained by this method correlated with the values of surface hydrophobicities obtained by conventional assay using a hydrophobic probe. Thus this measuring method using an SPR sensor and Triton X-100 is expected to be a tool for quantifying surface hydrophobicities of immobilized proteins.