Herein, we develop a novel method for designing electrochemical biosensors with both current and potential signal outputs for the simultaneous determination of two species in a living system. Oxygen (O2) and pH, simple and very important species, are employed as model molecules. By designing and synthesizing a new molecule, Hemin-aminoferrocene (Hemin-Fc), we create a single electrochemical biosensor for simultaneous detection and ratiometric quantification of O2 and pH in the brain. The reduction peak current of the hemin group increases with the concentration of O2 from 1.3 to 200.6 μm. Meanwhile, the peak potential positively shifts with decreasing pH from 8.0 to 5.5, resulting in the simultaneous determination of O2 and pH. The Fc group can serve as an internal reference for ratiometric biosensing because its current and potential signals remain almost constant with variations of O2 and pH. The developed biosensor has high temporal and spatial resolutions, as well as remarkable selectivity and accuracy, and is successfully applied in the real-time quantification of O2 and pH in the brain upon ischemia, as well as in tumor during cancer therapy.