An ultrasensitive electrochemical biosensor for the determination of hydroquinone (HQ) and chlorogenic acid (CGA) has been prepared by horseradish peroxidase (HRP) immobilization onto nanohydrogels made of laponite, gold nanoparticles (AuNP) and a vinylbenzyltriethylammonium polycation copolymerized with vinylbenzylthymine groups. The structure and active site of the enzyme were not modified upon immobilization, as determined by UV-Vis and FTIR spectroscopies. The biosensor showed remarkable electroanalytical properties for detection of HQ and CGA, e.g. linear stationary current up to 120 mu M and 4.2 mu M, limit of detection (LOD) of 1.6 +/- 0.2 nM and 2.7 +/- 0.1 nM for CGA, and sensitivities of 218 +/- 4 mu A.mM(-1) and 132 +/- 4 mu A.mM(-1), respectively. The electroanalytical capabilities of the biosensor was successfully tested in the quantification of the total polyphenol content in green coffee and yerba mate beverages, yielding equivalent results than those obtained with the classical Folin-Ciocalteu method. Nonetheless, our biosensor showed remarkable advantages due to its ultra-sensitivity, together with smaller sample volumes and shorter detection times required, improving its analytical application. (C) 2018 The Electrochemical Society.