A disposable paper-based bipolar electrochemical biosensor is reported for determination of glucose. The closed bipolar electrochemical cell is fabricated on a small part of paper using a laser printing-based process for paper hydrophobization. The bipolar and driving electrodes are provided by pressing the writing pencil HB on the paper. The mechanism of sensing of glucose is oxidation of the analyte in the sensing cell using glucose oxidase followed by reduction of the produced H2O2 by application of an external potential (10.0V). This causes the oxidation of K4Fe(CN)6 in the presence of Fe(II) ions and subsequent formation of Prussian Blue (PB) particles in the reporting cell. The intensity of the blue color in the reporting cell is used as a visual and colorimetric signal that can be digitally read using a scanner of digital camera. The parameters affecting the performance of the device were optimized using experimental design and chemometrics modeling. The P-BPE represents a very wide response range that extends from 0.1mmol.L-1 to 4.0mol.L-1 in the case of hydrogen peroxide, and from 0.1 to 50mmol.L-1 in the case of glucose. The limit of detections for hydrogen peroxide and glucose are 4.9μmol.L-1 and 70μmol.L-1 respectively. Graphical abstract Analyte solution (H2O2) and deionized water is injected to the sensing and the reporting cells respectively. By applying of an external potential, H2O2 reduction and potassium ferrocyanide (K4Fe(CN)6) oxidation is performed. This provides the appropriate condition for Prussian blue (PB) production (dark blue) in the reporting cell.