A new approach for the determination of bilirubin by an enzyme-based fiber-optic fluorescence biosensor is presented. Bilirubin is oxidized to biliverdin by bilirubin oxidase, and is quantitatively determined by measuring the fluorescence intensity change caused by the oxygen consumption in the enzymatic reaction. Bilirubin oxidase is immobilized onto an affinity membrane which is layered onto an optical indicating membrane. Two membranes are fixed around the common end of a bifurcated fiber-optic bundle. The limit of detection is 4.4 × 10−7M. The linearity of calibration curve is between 4.4 × 10−7M and 3.0 × 10−4M.