A glassy carbon electrode was modified by co-immobilization of tyrosinase and a conducting poly(1-vinylimidazole)-based osmium polymer on its surface. The ability of this biosensor to function as a detector for the HPLC determination of phenolic compounds, such as p-aminophenol, phenol, p-cresol, catechol and p-chlorophenol, was then assessed. The mobile phases investigated were binary mixtures of methanol and phosphate buffer. The effects of operational parameters, such as the concentrations of the electrode-modifying compounds, i.e., the osmium polymer and the cross-linking agent, poly(ethylene glycol), the mobile phase flow rate, the working potential and the biosensor's short-term stability were investigated and optimized. The biosensor exhibited a 100–200-fold improvement in sensitivity and detection limit when compared with a UV/VIS spectrophotometric detector. The amperometric phenol biosensor's was able to detect catechol, phenol, p-cresol, m-cresol and four other phenolic compounds in cigarette filter tips.