Nitroreductase (NR) was modified on glass carbon electrode and used in nitrobenzene analysis. Both cyclic voltammetry (CV) and triple potential step chronocoulometry (TPSC) with a narrow potential step interval were used. The modification of nitroreductase improved the sensitivity and selectivity of nitrobenzene detection. Both the peak area and peak current were examined for quantitative analysis, showing a linear relation between nitrobenzene concentration and peak area, and a logarithmic relation for peak current, with correlation coefficients of 0.9981 and 0.9993, respectively. Statistical tests to evaluate the reliability of TPSC showed that there was no evidence of systematic error, and it was not significantly different from CV or high performance liquid chromatography (HPLC). The new method could avoid most interferents, showing good selectivity.