In this research, we have demonstrated a one-step electrochemical deposition of dendritic gold nanostructures (DGNs) on a graphite rod (GR) electrode without any template, seeds, surfactants, or stabilizers. Three electrochemical methods, namely, constant potential amperometry (CPA), pulse amperometry, and differential pulse voltammetry, were used for DGN synthesis on GR electrode and further application in enzymatic glucose biosensors. Formed gold nanostructures, including DGNs, were characterized by a field emission scanning electron microscopy. The optimal concentration of HAuCl4 (6.0 mmol L−1), duration of DGNs synthesis (400 s), electrodeposition potential (−0.4 V), and the best electrochemical method (CPA) were determined experimentally. Then the enzyme, glucose oxidase, was adsorbed on the surface of DGNs and covalently cross-linked with glutaraldehyde vapor. The enzymatic glucose biosensor based on DGNs electrodeposited at optimal conditions and modified with glucose oxidase showed a quick response (less than 3 s), a high saturation current (291 μA), appropriate linear range (up to 9.97 mmol L−1 of glucose, R2 = 0.9994), good repeatability (RSD 2.4, 2.2 and 1.5% for 2, 30, 97 mmol L−1 of glucose), low limit of detection (0.059 mmol L−1, S/N = 3) and good stability. Additionally, this biosensor could be successfully applied for glucose determination in real samples with good accuracy. These results proved the principle of enzymatic glucose biosensor development based on DGNs as the basis for further investigations.