Nitrite is a multipurpose marker that needs to be detected quickly and accurately, including for water pollution and human urinary tract infections. In this study, we reported a novel nitrite-reducing bacteria electrochemical biosensor based on the metronidazole-treated biocathode (MT-NBEB). The biocathode with high nitrite response sensitivity used was first prepared by polarity inversion method and then treated with metronidazole to selectively remove the interfering electroactive bacteria for selectivity improvement. MT-NBEB could detect nitrite in the range of 0.0001 mg NO2−-N L−1–8 mg NO2−-N L−1 within 1.7 min and maintain stable detection performance for over 50 continuous cycles with relative standard deviations < 2.4%. Besides, the response signals of MT-NBEB were not affected by the common inorganic salts (such as nitrate and ammonia) and organic matter (such as acetate). MT-NBEB successfully detected nitrite in five types of wastewaters with relative errors < 14.3%. Our study provided a feasible way to prepare highly selective and sensitive electrochemical biosensors to quickly and accurately detect nitrite in real wastewaters.