Co-immobilization of low molecular weight mediators and glucose oxidase in polyelectrolyte membranes results in glucose test strips operating in millimolar concentration range. Density and charge of polyelectrolyte membranes formed on the surface of the screen-printed electrodes allow to control the diffusion of mediators. Negatively charged perfluorosulfonated ionomer (PFSI) hampers the diffusion of the commonly used ferricyanide (III) ion, while the hexammine ruthenium (III) cation apparent diffusion coefficient in PFSI membrane remains the same as without the membrane. In contrast to PFSI, electrode modification with positively charged chitosan leads to additional adsorption of potassium hexacyanoferrate on the membrane. Additionally, the rate of mediator leakage from the membrane was found to govern the sensitivity of the resulting biosensors. The leakage rate also depends on the density and charge of the polyelectrolyte and mediator. However, the main advantage of the proposed simple approach of single-step deposition of three-component membrane-forming mixtures on the screen-printed electrodes is the extended upper limit of the linearity: 30-50 mM glucose. Hence, the obtained test strips are suitable for glucose detection in undiluted blood.