Biosensors based on hydrophobic ionic liquids (ILs) derived from the bis(trifluoromethylsulfonyl)imide [(CF(3)SO(2))(2)N(-) = Tf(2)N(-)] anion associated with three different imidazolium cations: 1-butyl-3-methylimidazolium (BMI x Tf(2)N), 1-decyl-3-methylimidazolium (DMI x Tf(2)N) and 1-tetradecyl-3-methylimidazolium (TDMI x Tf(2)N), along with laccase from Aspergillus oryzae, were constructed and optimized for determination of rutin. The laccase catalyzes the oxidation of rutin to the corresponding o-quinone, which is electrochemically reduced back to rutin. The best performance was obtained with 50:20:15:15% (w/w/w/w) as the graphite powder:laccase:Nujol:ILs composition in 0.1 mol L(-1) acetate buffer solution (pH 5.0). The parameters for the square-wave voltammetry experiments and scanning electron microscopy images of the biosensors were studied. Under the selected conditions, the cathodic peak current increased linearly in the rutin concentration ranges of 4.77x10(-6) to 4.62x10(-5) mol L(-1), 5.84x10(-6) to 5.36x10(-5) mol L(-1) and 5.84x10(-6) to 5.36x10(-5) mol L(-1) using the (I) BMI x Tf(2)N-laccase, (II) DMI x Tf(2)N-laccase and (III) TDMI x Tf(2)N-laccase, respectively. The rutin contents of commercial samples of pharmaceuticals were successfully determined by the biosensors and the results compared well with those obtained using the official method. The studies on rutin recovery from these samples gave values of 96.9-104.6%.