This work reports the fabrication of fully drawn fluidic electrochemical paper-based analytical devices (ePADs) using a low-cost and rapid dual-step pen-on-paper plotting approach for the enzymatic assay of glucose. The desired fluidic patterns were formed on the paper substrate using an inexpensive computer-controlled x-y plotter and a commercial hydrophobic marker pen. Electrodes were further deposited on the devices using a second x-y plotting step with a commercial writing pencil. Enzymatic paper-based biosensors were prepared by immobilizing glucose oxidase and potassium ferricyanide (as a mediator) at the test area of the ePADs; the cost of each biosensor was < 0.05 € in terms of materials. A simple, low-cost and rapid method was further developed for the amperometric enzymatic determination of glucose using the biosensing ePADS. The limit of quantification was 0.08 mmol L−1, the between-device reproducibility (expressed as the % relative standard deviation of the response at 6 different ePADs) was < 12% and the assay time was 120 s. The ePADs were applied to the determination of glucose in food samples with recoveries in the range 94–106%.