Novel and effective biosensors based on Ag or Au nanoparticles dispersed in ionic liquid (IL) 1-butyl-3-methylimidazolium hexafluorophosphate (BMI·PF₆) and laccase (Lac) from Aspergillus oryzae immobilized in chitosan (Chi) chemically cross-linked with cyanuric chloride (CC) were constructed. This enzyme catalyzes the oxidation of luteolin to the corresponding o-quinone, which is electrochemically reduced back to luteolin at 0.35 V vs.Ag/AgCl. Square-wave voltammetry was used for the electrochemical determination of luteolin at the Lac-nanoparticles-BMI·PF₆ biosensors. The best performance was obtained with 50:20:15:15% (w/w/w/w) as the graphite powder:Chi-CC:Nujol:Ag-BMI·PF₆ or Au-BMI·PF₆ composition (Lac 0.29 units mL⁻¹) in 0.1 M acetate buffer solution (pH 4.0) with frequency, pulse amplitude and scan increment at 50 Hz, 100 mV, and 5.0 mV, respectively. Under optimized conditions, the cathodic currents increased linearly for the luteolin concentration range of 0.099–5.825 µM with detection limits of 0.054 ± 0.004 µM (Ag-BMI·PF₆) and 0.028 ± 0.002 µM (Au-BMI·PF₆). These biosensors demonstrated high sensitivity, good repeatability and reproducibility, and long-term stability (13% decrease in response over 70 days). The recovery study for luteolin in chamomile tea samples gave values of 91.8–104.8%. The influence of Lac immobilized in Chi-CC and nanoparticles-BMI·PF₆ contributes to the excellent performance of the biosensors.