Fiber-optic biosensors have been studied intensively because they are very useful and important tools for monitoring biomolecular interactions. Here we describe a fluorescence detection fiber-optic biosensor (FD-FOB) using a sandwich assay to detect antibody-antigen interaction. In addition, the quantitative measurement of binding kinetics, including the association and dissociation rate constants for immunoglobulin G (IgG)/anti-mouse IgG, is achieved, indicating 0.38 x 10(6) M(-1) s(-1) for k(a) and 3.15 x 10(-3) s(-1) for k(d). These constants are calculated from the fluorescence signals detected on fiber surface only where the excited evanescent wave can be generated. Thus, a confined fluorescence-detecting region is achieved to specifically determine the binding kinetics at the vicinity of the interface between sensing materials and uncladded fiber surface. With this FD-FOB, the mathematical deduction and experimental verification of the binding kinetics in a sandwich immunoassay provide a theoretical basis for measuring rate constants and equilibrium dissociation constants. A further measurement to study the interaction between human heart-type fatty acid-binding protein and its antibody gave the calculated kinetic constants k(a), k(d), and K(D) as 8.48 x 10(5) M(-1) s(-1), 1.7 x 10(-3) s(-1), and 2.0 nM, respectively. Our study is the first attempt to establish a theoretical basis for the florescence-sensitive immunoassay using a sandwich format. Moreover, we demonstrate that the FD-FOB as a high-throughput biosensor can provide an alternative to the chip-based biosensors to study real-time biomolecular interaction.