A new approach to the production of optical fiber sensors is described which is based on a dynamic modification procedure. In this approach, the optical fiber surface is rendered hydrophobic through the covalent attachment of a C18 moiety. Specific sensing ligands are then associated with this surface through either their inherent or designed hydrophobicity. To investigate the feasibility of the approach, an optical fiber sensor has been constructed for riboflavin binding protein in which 3-octylriboflavin is associated with the fiber surface. Fluorescence quenching occurs upon binding of the protein to the immobilized riboflavin derivative. The sensor possesses a minimum measurable quantity of 7.3 pmol of binding protein in a probe volume which is less than 10 microL. With this approach, the sensing surface was repetitively regenerated 15 times over a 1-h period with less than a 5% variation in sensor-to-sensor performance. The approach is general, and with minor variations it can be used in a variety of sensing situations.