A fiber optic biosensor was constructed from Pleurotus ostreatus laccase for the detection of polyphenolic compounds. Laccase was immobilized on the surface of the commercially available fiber optic oxygen sensor spots by using 3-aminopropylsilanetriol, glutaraldehyde, and amino-modified carboxycellulose. A diffusion layer containing tetramethyl orthosilicate (TMOS), trimethoxymethylsilane (Tri-MOS), and polyvinyl alcohol (PVA) was added to the immobilized laccase layer. The consumption of oxygen as a result of laccase activity was monitored using a fiber optical measuring setup with catechol as a model substrate. The optimal enzyme amount was determined as 1.5 mg per 50 µL of enzyme layer mixture, and with one diffusion layer and at pH 6.9, optimum detection conditions were attained. The biosensors have high reproducibility, stability (at least 85 days if stored in PBS at 4 °C), and convenient measurement duration (ca. 25 min between two successive measurements). The biosensor was found to have a broad linear working range for catechol (40-600 μM) and to be applicable to a flow-through system. In summary, an easy-to-produce, reproducible, and stable laccase sensor with a broad linear working range was produced. The sensor has potential in the food industry as well as in environmental monitoring for the detection of phenolic compounds.