—Hybrid thin films based on Hydrocalu-mite (Ca 2 AlCl layered double hydroxide LDH) and tyrosinaseenzyme have been used for the elaboration of a high sensitive amperometric biosensor detecting polyphenols extracted from green tea. Structural properties of LDH nanomaterials were characterized by X-ray powder diffraction and Infra-Red spectroscopy, confirming its crystalline phase and chemical composition. Ca 2 AlCl-LDHs-thin films were deposited by spin-coating, and studied by atomic force microscopy to obtain information about the surface morphology of this host matrix before and after enzyme's immobilization. Electrochemical study using cyclic voltammetry and chronoamperometry shows good performances of the built-in biosensor with a high sensitivity for polyphenols concentrations ranging from 24 pM to 2.4 µM and a limit of detection of 1.2 pM. Index Terms—Amperometric biosensor, layered double hydroxide, tyrosinase, hybrid nanomaterial, atomic force microscopy.