In the present work, we have studied the impact of surface charge on the peroxidase mimetic activity of gold nanorods (GNRs). Gold nanorods with positive (cetyltrimethylammonium bromide (CTAB)) and negative (poly (styrene sulfonic acid) (PSS)) surface charges were synthesized and subsequently characterized by UV-visible spectroscopy, zeta potential analysis, and transmission electron microscopy (TEM). The two kinds of gold nanorods were evaluated for their peroxidase mimetic activity. Gold nanorods with different surface charges can able to mimic the enzyme activity near acidic pH over a wide range of temperatures. Poly (4-styrene sulfonic acid)-stabilized negative gold nanorods (N-GNRs) possessed higher peroxidase mimetic activity compared to CTAB-stabilized positive GNRs (P-GNRs) when tested with a low concentration of tetramethylbenzidine (1 mM) and hydrogen peroxide (0.1 M). The significant impact of surface charges of GNRs on the catalytic reactions may help us to make a prototype in creating nanozymes as peroxidase mimics.