Bipolar electrochemistry (BPE) has been lately explored as a simple, reliable and novel electrochemical technique for the adjustment of various conductive substrates. Herein, BPE is performed to derive both of cathode and anode electrodes for the development of mediatorless/membraneless biofuel cell (BFC). On one hand, a preferable substrate for immobilization of bilirubin oxidase enzyme is prepared based on the electropolymerization of thiophene-3-carboxcylic acid (TCA) on an Au microflm as a bipolar electrode. The resulted biocathode as novel bioelectrocatalyst ofers a high electrocatalytic activity toward direct oxygen reduction reaction (ORR) with onset potential and current density of 0.55V (vs. Ag/AgCl) and 867μA cm−2, respectively. On the other hand, another analogous Au bipolar electrode is electroplated through BPE to derive Au nanostructures (AuNSs). This modifed Au electrode is utilized as an anodic platform for immobilization of favin adenine dinucleotide-dependent glucose dehydrogenase (FAD-GDH) enzyme aimed at electrocatalytic glucose oxidation. The prepared bioanode displays a current density of 2.7mAcm−2 with onset potential of −0.03V. Finally, the proposed bioanode and biocacthode in an assembled membraneless glucose/O2 BFC ofers a power output of 146μW cm−2 with open circuit voltage of 0.54V. This novel BPE method provides disposable electrochemical platforms for design of novel sensors, biosensors or other devices.