A portable biofuel cell with an anode modified with glucose oxidase/ferrocene and a cathode modified with bilirubin oxidase was fabricated on a flexible polyimide substrate. The anode and the cathode with an area of 3×10 mm² were separated with a gap of 1 mm. Solidified glucose biofuel units were prepared by solidifying 50–200 mM glucose aqueous solution containing 0.5–2.5% agarose. The influences of the biofuel volume and glucose concentrations on power generation were investigated. The maximum power density was almost independent on the agarose concentration, and it continuously decreased as time clasped possibly due to the consumption of glucose and/or release of the enzymes from the electrodes. The maximum power and power density were 0.32 μW and 1.08 μW/cm² at 0.17 V, respectively when the solidified hydrogel biofuel unit with a dimension of 13×24×4 mm³ containing the 100 mM glucose aqueous solution and 2.5 wt% agarose was used. The biofuel cell continued to deliver the power density over 0.5 μW/cm² for more than 1 h.