We have developed a technique to immobilize a multilayer enzyme assembly on an optical fiber surface. The technique utilizes a bifunctional amino coupling agent to link between different layers of enzymes. The method offers the flexibility of controlling the number of enzymes on a fiber surface. Multilayers of enzyme — Alkaline Phosphatase have been characterized using ellipsometry and surface plasma resonance. Chemiluminecence experiments have been conducted as a function of number of enzyme layers. The results indicated that at least 3 layers of enzyme can be assembled on a fiber surface. Utilising the new immobilization technique, a novel fiber optic biosensor system for the detection of organophosphorous-based pesticide has been developed. The detection mechanism is the pesticide inhibition of alkaline phosphatase catalyzed chemiluminescence. As a model analyte, paraoxon with ppb level concentration has been detected.