An ultraviolet pulsed laser ablation of polyimide film coated with platinum has been used to enhance the sensitivity for the application as an electrochemical biosensor. Densely packed cones are formed on polyimide surface after UV irradiation which results in increase of surface area. In order to apply the sensitivity improvement of laser ablated polyimide film electrodes, the glucose oxidase modified biosensor was fabricated by using an encapsulation in the gel matrix through sol-gel transition of tetraethoxysliane on the surface of laser ablated polyimide film. The optimum conditions for glucose determination have been characterized with respect to the applied potential and pH. The linear range and detection limit of glucose detection were from 2.0 mM to 18.0 mM and 0.18 mM, respectively. The sensitivity of glucose biosensors fabricated with laser ablated polyimide film is about three times higher than that of plain polyimide film due to increase in surface area by laser ablation.