The objective of this study was to develop a ferrocene mediated glucose biosensor for reverse iontophoresis. An amperometric ferrocene mediated glucose biosensor based on a three electrodes planar configuration was constructed using screen printing technique. Different combinations of glucose oxidase and ferrocene loading were drop coated onto the surface of the amperometric transducer. The amperometric transducer was characterized electrochemically using cyclic voltammetry and its electrochemical characteristics (ΔE p = 70 mV, I pa/I pc = 0.89) were found close to an ideal amperometric transducer. The biosensor on the detection of glucose at 200 mV (vs. Ag/AgCl) showed a linear response range (0–4 mM). The response time of the biosensor was about 10 s. Finally, the biosensor was used together with reverse iontophoresis technique. By the use of an actual model for evaluation, an excellent linear relationship (r 2 = 0.99) was found between the glucose concentration of the actual model and the biosensor current response. In conclusion, a ferrocene mediated glucose biosensor incorporated with reverse iontophoresis function was developed.