A novel electrochemical biosensor for the determination of pyrogallol (PG) and hydroquinone (HQ) has been constructed based on the poly L-arginine (poly(L-Arg))/carbon paste electrode (CPE) immobilized with horseradish peroxidase (HRP) and silver nanoparticles (AgNPs) through the silica sol-gel (SiSG) entrapment. The electrochemical properties of the biosensor were characterized by employing the electrochemical techniques. The proposed biosensor showed a high sensitivity and fast response toward the determination of PG and HQ around 0.18 V. Under the optimized conditions, the anodic peak current of PG and HQ was linear with the concentration range of 8 mu M to 30 x 10(-5) M and 1-150 mu M. The limit of detection (LOD) and limit of quantification (LOQ) were found to be 6.2 mu M, 20 mu M for PG and 0.57 mu M, 1.92 mu M for HQ respectively. The electrochemical impedance spectroscopy (EIS) studies have confirmed that the occurrence of electron transfer at HRP-SiSG/AgNPs/poly(L-Arg)/CPE was faster. Moreover the stability, reproducibility and repeatability of the biosensor were also studied. The proposed biosensor was successfully applied for the determination of PG and HQ in real samples and the results were found to be satisfactory. (C) 2013 Elsevier Inc. All rights reserved.