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We present a new and fast method for preparation of ultrathin layers of biomolecules on solid substrates and its application as a useful tool for sensing biomolecules in an affinity biosensor. Nonamphiphilic trimethylsilyl ether−cellulose monolayers are used as interfaces to reduce nonspecific interactions between solid surfaces and biomolecules. These films are transferred on optical waveguides by the Langmuir−Blodgett technique. After their stabilization by cleavage of the hydrophobic trimehylsilyl groups, the cellulose films serve as excellent matrixes for the immobilization of proteins at high density. Cyanurchloride serves as a cross-linker between the cellulose layer and the proteins. The activity and biological specificity of these interfaces is controlled by an enzymatic assay and direct sensing by an evanescent wave immunosensor.