This paper presents a novel multilevel membraneless enzymatic biofuel cell. For the first time, we introduce the use of thin polyester films as flexible electrode substrates for a Glucose/O2 microfluidic biofuel cell. More specifically, we report a proof-of-concept based on a three dimensional (3D) microfluidic chip fabricated via rapid prototyping where two T-shaped microchannels are vertically stacked one above the other. Via the integration of two in-reservoirs, only three access ports are required and an even repartition of laminar flows is ensured within each microchannel. Both channels have gold electrodes patterned on their top and bottom walls. With four anodes/four cathodes having an individual area of 25mm2, our structure is only 425μm thick. Enzymatic reactions occur via the use of glucose oxidase and hexacyanoferrate flowing in the anolyte solution whereas freely suspended laccase and 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) are introduced in the catholyte stream. Under a flow rate of 150μLmin−1 and with all anodes/cathodes connected in parallel, our structure can generate a maximum net power of 12.5±0.05μW. Connected to a voltage boost converter increasing the nominal output voltage to 3.1V, we demonstrate that our proof-of-concept can already be exploited to supply electrical energy to a wireless sensor sending temperature measurements to a remote computer.