Hybrid materials containing organic and inorganic structures are very important because they have the advantages of each of their components. Intercalation of the organic molecule to inorganic structures such as clay allows the preparation of multifunctional new immobilization matrices that contain both the strength properties of the clays and the properties of the organic molecule such as its functional groups (amine, carboxyl or hydroxyl etc.). In this study, 4-aminothiophenol intercalated montmorillonite (4ATP-Mt) was prepared and used as an immobilization layer for the pyranose oxidase (PyOx) enzyme on a glassy carbon (GC) electrode. Firstly, 4ATP intercalation of Mt was carried out, and the success of intercalation was proven using Fourier-Transform Infrared Spectroscopy (FTIR), X-ray Diffractometer (XRD), and Thermogravimetric Analysis (TGA) techniques. Then PyOx was immobilized using 4ATP-Mt, BSA and glutaraldehyde as a cross-linker on the surface of GC electrodes. To show the coating steps for the preparation of 4ATP-Mt/PyOx biosensors, cyclic voltammetry (CV), differential pulse voltammetry (DPV) and electrochemical impedance spectroscopy (EIS) techniques were applied, and surface morphology was visualized by scanning electron microscopy (SEM). After optimization of working conditions, analytical performance parameters were determined for glucose detection. The 4ATP-Mt/PyOx biosensor exhibited a wide linear concentration range between 0.01 and 0.5 mM (LOD: 1.0 mu M) for glucose. Additionally, the fabricated 4ATP-Mt/PyOx was assayed for the analysis of glucose in artificial body fluids and drinks.