Humic acid (HA) is a natural source of organic matter obtained from the soil. It is a stable and water-soluble compound with heterogeneous organic components which helps the plants to absorb water and other nutrients from the soil. HA contains quinoid redox active groups for electron transfer reactions. In this work, we have used HA as a dispersing agent to prepare Halloysite nanotube (HNT) dispersion. HNT is a one-dimensional, layered aluminosilicate nanotube which contains hydroxyl groups on the surface. HNT has several unique properties such as high surface area, mesoporous material, presence of polar charges and low-cost. As-prepared HA/HNT dispersion was characterized using Ultraviolet-Visible spectroscopy (UV-Vis), X-Ray Diffraction (XRD), and High-resolution transmission electron microscopy (HR-TEM). Furthermore, HA/HNT dispersion was used to modify a glassy carbon electrode (GCE) for successful immobilization of flavin adenine dinucleotide (FAD) from the deoxygenated phosphate buffered saline (PBS, pH 7.4) solution containing FAD molecules. Electrochemical properties of FAD immobilized on GCE/HA/HNT (GCE/HA/HNT/FAD) were studied by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The GCE/HA/HNT/FAD biosensor showed a redox peak centered at (E-o') -0.45 V in PBS (pH 7.4) at a scan rate of 50 mV/s. This new FAD (electron transfer mediator) based biosensor showed good electrocatalytic activity towards the reduction of hydrogen peroxide (H2O2) at -0.48 V. The calibration graph was obtained from 1 to 250 mu M of H2O2 and the limit of detection (LOD) was found to be 0.49 mu M. The interference study was also performed in the presence of uric acid (UA), dopamine (DA), ascorbic Acid (AA) and oxalic acid (OA) which confirmed that these molecules did not interfere in the electrochemical measurements. Moreover, the GCE/HA/HNT/FAD biosensor exhibited high reproducibility and repeatability during electrochemical analysis. The real applicability of the biosensor was also demonstrated by detecting H2O2 in an antiseptic solution.