Ascorbic acid (AA), dopamine (DA), and uric acid (UA) are biological molecules coexisting in human serum. AA plays a vital role in regulation of the redox meta bolism, promoting the synthesis of catecho lamine and scavenging reactive oxygen species (ROS). [1] DA controls central nervous system [2,3] and cardiovascular system functions. [4][5][6] Low level of DA is closely related to diseases such as Parkinson's and Alzheimer's disease. [7] UA is the final metabolite of purine and sev eral diseases such as gout, renal disorders, cardiovascular, etc. often result from exces sive uric acid level. [8] Thus, monitoring the concentration level of these biomolecules in human body metabolism is vital for healthcare and clinical diagnostics. Electro chemical sensor has been widely applied in medical, food, pharmaceutical, environ ment fields for analytes detecting (e.g., bio markers, toxic chemicals, drugs). [9][10][11] This technique has been considered to be supe rior to other detecting methods such as highperformance liquid chroma tography, Uv-vis spectrometry and chemiluminescence due to its low cost, simple equipment, and ease of operation. [12] As the development of portable health care devices (e.g., wearable biosensor, point of care sensing plat form, etc.), electrochemical sensor has become an idea candi date to realize those devices since it can be easily integrated on an electrical circuit. However, to meet the demand of practical application, it is still challenging to develop an electrochemical sensor with high selectivity and sensitivity. AA, DA, and UA are electrochemically active biomolecules and can be oxidized under different applied potential in an electrochemical system. [13,14] Theoretically, as to the different molecular structure of AA, DA, and UA, they are supposed to show volt-ampere responses at distinguishable oxidation potentials. However, since their oxi dation potentials are too close, an overlapping potential usually occurs when simultaneously detect AA, DA, and UA in a ternary mixture at a bare electrode, resulting in a low resolution or even fail to distinguish each signal. Thus, electrode modifying is cru cial for AA, DA, and UA detection. Herein, a novel electrochemical biosensor is simply constructed by using graphene encapsulated CoFe alloy nanocomposite (CoFe@G) as the electrode modifier for highly sensitive and simultaneous detection of ascorbic acid (AA), dopamine (DA), and uric acid (UA). Cyclic voltammetry tests show well-separated peaks for AA at −7 mV, DA at 186 mV, and UA at 325 mV. CoFe@G modifier improves the differentiation of oxidative potential gap between AA and DA, realizing selective detection of these biomolecules. The mechanism of selective detection ability is studied by electrochemical impedance spectroscopy, quartz crystal microbalance (QCM), and in situ attenuated total reflection surface-enhanced infrared absorption spectroscopy (ATR-SEIRAS) analysis. It is demonstrated that modifying CoFe@G on glassy carbon electrode (GCE) can effectively lower the oxidation pot...