In this article, a bare glassy carbon electrode (GCE) surface was directly activated by a simple in situ chemical method, which was characterized by scanning electron microscopy (SEM), atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS) and cyclic voltammetry (CV), differential pulse voltammetry (DPV) and electrochemical impedance spectroscopy (EIS). Based on these results, it was found that oxygen-containing functional groups at the modified GCE surface were enhanced with a low damage to the surface state. Hence, the modified GCE exhibited an excellent performance, such as the negatively charged surface, good reproducibility and high selectivity. The resulting electrode was applied as a sensitive sensor for detection of antioxidant tertiary butyl hydroquinone (TBHQ), and a good linear relationship was obtained between the oxidation peak current and the concentration in a broad range of 1.0 μM–1.1 mM, with detection limits of 67 nM (S/N = 3) by DPV. Electrochemical parameters of TBHQ on the resulting GCE were also investigated, suggesting that the modified GCE could promote electron transfer kinetics towards the electrochemical reaction of TBHQ. Besides, the present method was used for determination of TBHQ in jatropha biodiesel with recovery ranging from 95.2% to 103.2%.