A new reagentless biosensor for the square-wave voltammetric determination of rutin in pharmaceutical formulations was developed by immobilization of gilo (Solanum gilo) crude extract in chitosan matrix. The gilo tissue acts as a source of peroxidase. The highest biosensor performance was obtained after immobilization of the peroxidase in chemically crosslinked chitosan with epichlorohydrin and glutaraldehyde that was incorporated in a carbon paste electrode. In the presence of hydrogen peroxide this enzyme catalyses the oxidation of rutin to quinone and the electrochemical reduction of the product was obtained at a fixed potential of +124 mV versus Ag/AgCl (3.0 M KCl). The performance and factors influencing the resulting biosensor were studied in detail. The bioelectrode exhibited a linear response for rutin concentrations from 3.4 × 10−7 to 7.2 × 10−6 M (r = 0.9998) and the recovery of rutin from the samples ranged from 96.2 to 102.4%. The detection and quantification limits were 2.0 × 10−8 and 6.3 × 10−8 M, respectively. The relative standard deviation was less than 1.0% for solutions containing 3.4 × 10−7 to 7.2 × 10−6 M rutin in 0.1 M phosphate buffer solution at pH 7.0 (n = 10). The lifetime of this biosensor was 8 months (at least 500 determinations).