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An electrochemical sensor based on a glassy carbon electrode (GCE) modified by three-dimensional graphene (3D-GE) and polyglutamic acid (PGA) was developed to quantitatively detect Amoxicillin (AMX), a worldwide used antibiotic. AMX response at PGA/3D-GE/GCE involving the transfer of one electron and an equal number of protons were determined using electrochemical approaches. Density functional theory simulations were performed to give insights on the reaction mechanism of AMX on the surface of the modified electrode. When the optimization of the experimental conditions was completed, the linear range of AMX was 2–60 μM. Besides, the detection limit was calculated as 0.118 μM (S/N = 3). And the modified electrode could detect the concentration of the AMX in human urine samples. Overall, the developed PGA/3D-GE/GCE for determination of AMX shows great potentials in practice. Image 1 • A friendly and sensitive method for the determination of amoxicillin was developed. • Polyglutamic acid/three-dimensional porous graphene (PGA/3D-GE) was used for the modification of electrode. • A new reaction mechanism of amoxicillin was proposed by combining computational simulation with electrochemical methods. [ABSTRACT FROM AUTHOR]