The design of a new tyrosinase biosensor with improved stability and sensitivity is reported. The biosensor design is based on the construction of a graphite-Teflon composite electrode matrix in which the enzyme and colloidal gold nanoparticles are incorporated by simple physical inclusion. Experimental variables such as the colloidal gold loading into the composite matrix, the enzyme loading and the potential applied to the bioelectrode were optimized. The Tyr-Au(coll)-graphite-Teflon biosensor exhibited suitable amperometric responses at -0.10 V for the different phenolic compounds tested (catechol; phenol; 3,4-dimethylphenol; 4-chloro-3-methylphenol; 4-chlorophenol; 4-chloro-2-methylphenol; 3-methylphenol and 4-methylphenol). The limits of detection obtained were 3 nM for catechol, 3.3 microM for 4-chloro-2-methylphenol, and approximately 20 nM for the rest of phenolic compounds. The presence of colloidal gold into the composite matrix gives rise to enhanced kinetics of both the enzyme reaction and the electrochemical reduction of the corresponding o-quinones at the electrode surface, thus allowing the achievement of a high sensitivity. The biosensor exhibited an excellent renewability by simple polishing, with a lifetime of at least 39 days without apparent loss of the immobilized enzyme activity. The usefulness of the biosensor for the analysis of real samples was evaluated by performing the estimation of the content of phenolic compounds in water samples of different characteristics.