The enantioselective interaction between horseradish peroxidase (HRP) and arginine enantiomers was investigated by electrochemical methods through studying the electrocatalytic activity of H2O2 biosensor, which was obtained through l-arginine or d-arginine functionalized multi-walled carbon nanotubes (d-Arg-MWCNTs or l-Arg-MWCNTs) immobilizing horseradish peroxidase (HRP) on glassy carbon electrode. Cyclic voltammetric and chronoamperometry were used to characterize the properties of the biosensor. Under the optimal conditions, LAM-CS@HRP/dpAu/GCE biosensor showed better electrocatalytic activity to H2O2 compared to DAM-CS@HRP/dpAu/GCE and MWCNTs-CS@HRP/dpAu/GCE, implying that the different configurations of nanocomposites have different interactions with HRP. The currents of LAM-CS@HRP/dpAu/GCE biosensor had a linear relationship with the concentration of H2O2 in the range of 2.5×10(-6) to 2.9×10(-3)M with a detection limit of 8.3×10(-7)M (S/N=3). For MWCNTs-CS-HRP/dpAu/GCE electrode, the calibration range of H2O2 was from 6.4×10(-4) to 2.9×10(-2)M and a detection limit of 2×10(-5)M (S/N=3). For the case of DAM-CS@HRP/dpAu/GCE, there has a linear relationship with the concentration of H2O2 from 1.8×10(-5) to 2.6×10(-3)M and the detection limit is 6×10(-5)M (S/N=3).