A self-contained fiber-optic immunosensor was developed to measure the 16,500-Da protein myoglobin. The sensing element was constructed by entrapment of Cascade Blue-labeled antibody within polyacrylamide gel at the distal face of an optical fiber 300 microns in core diameter. The polyacrylamide gel composition was optimized to allow diffusion of myoglobin but to exclude hemoglobin and higher-molecular-mass proteins from the sensing area. The analytical signal was derived from fluorescence energy transfer between Cascade Blue and the heme group of myoglobin. Fluorescence quenching occurred when myoglobin bound to labeled antibody. The total amount of fluorescence quench was dependent on the antibody labeling conditions and the amount of antibody incorporated in the sensor gel matrix. Myoglobin concentrations > 5 nmol/L (83 micrograms/L) were measurable with response times of 15 to 130 min limited by diffusion into the sensing element. This report demonstrates the technical feasibility for a self-contained immunosensor to measure a protein analyte.