To enhance the reduction of Chlorpyrifos (CP) in farmland drainage, zero valent iron (ZVI) was introduced into the anaerobic bioreactor through batch experiments. Compared to the mono-cell system, the coupled bio-ZVI system showed better performance on the removal of CP. The optimal operating conditions were determined to be 1 g L-1 of iron shavings, 800 mu g L-1 of the initial CP concentration and 35 degrees C by comparing different ZVI types, ZVI dosages, initial CP concentrations and temperature. CP removal efficiency of 95.94% and 79.97% was obtained in the coupled bio-ZVI and control system under the optimal conditions, respectively. Scanning electron microscopy showed that iron shavings could enhance the granulation of the anaerobic sludge. Moreover, the relative abundances of species analyzed by high-throughput sequencing in the coupled system were higher than that in the control system. Especially, the highly enrichment of microorganisms capable of degrading CP or organophosphorus pesticide such as Cyanobacteria and Acinetobacter in the ZVI-amended system was detected. The main metabolite 3,5,6-trichloro-2-pyridinol (TCP) transformed from CP in the control system was higher than that in the coupled system, with the maximum levels of 253.84 mu g L-1 and 221.71 mu g L-1, respectively. Besides, TCP in the coupled system can be degraded more significantly, which could suggest that the presence of ZVI could facilitate the resistance of the biomass to the toxicity of TCP.