Fast and sensitive devices for monitoring of glucose are crucial in today’s medicine. Most of the commercial glucometers are currently based on enzymatic catalysis which provides reasonable sensitivity but has a drawback in limited enzyme stability. With the development of novel surfaces, new generation of non-enzymatic sensors characterized by high sensitivity and stability is emerging. In this work, gelatin-templated nanostructured gold electrodes were developed and applied for non-enzymatic glucose determination. The analysis was done by direct electrochemical oxidation during cyclic voltammetry and amperometry with the limit of detection at 1.3 μM. The ability to detect physiological levels glucose in presence of interferents was demonstrated on deproteinized human blood serum.