We report one-step synthesis of carbon-based microstructures on the surface of polyimide by using the 2D direct laser writing technique. It was shown that the properties of obtained conductive structures can be tuned by varying the conditions of the laser synthesis (scanning speed, laser power). The fabricated materials exhibited high electrochemical activity towards d-glucose measured by cyclic voltammetry analysis (limit of detection—0.564 μM and 6.31 μM for different linear ranges). Comparatively high sensing activity of the synthesized structures makes them promising materials for flexible non-enzymatic biosensing devices.