Microbial electrolysis cell (MEC) has been constructed to study the degradation characters of 2-chloro-4-nitrophenol (2C4NP) in waste water. The effects of applied voltage, initial concentration of substrate and co-matrix species on the reduction and degradation of 2C4NP were studied. Qualitative and quantitative analysis of 2C4NP residues and degradation intermediate by using UV-Vis, HPLC, HPLC/MS/MS, IC and other analytical testing techniques. The degradation mechanism of 2C4NP in MEC cathode was proposed. The results showed that electron and electroactive microorganisms would produce coupling effect and accelerate the degradation of 2C4NP under adding 0.5 V DC; Under the condition of satisfying the C/N ratio of electroactive anaerobic microorganism, the addition of organic substances such as glucose and sodium acetate which were easily degraded by microorganisms would hinder the degradation of 2C4NP in the cathode compartment. 2C4NP can be effectively degraded by adding appropriate amount of glucose as carbon source with the low C/N. 2C4NP undergoes reduction, dechlorination, denitrification and assimilation in the cathode compartment to form 2-chloro-4-aminophenol, 4-aminophenol, 2-chlorophenol, 2-chloro-4-hydroxyphenol, nitrophenol, hydroquinone, 4-hydroxyhexadienoic acid semialdehyde, valeric acid, oxalic acid and many other intermediate products. According to the degradation intermediates, the degradation mechanism of 2C4NP in the cathode compartment was presumed.