Increasing the value of food waste through anaerobic digestion is an attractive strategy. Meanwhile, the anaerobic digestion of kitchen waste also faces some technical challenges. In this study, four EGSB reactors were equipped with Fe-Mg-chitosan bagasse biochar at different locations, and the reflux pump flow rate was increased to change the upward flow rate of the reactor. The effects of adding modified biochar at different locations under different upward flow rate on the efficacy and microecology of anaerobic reactors treating kitchen waste were investigated. Results showed that Chloroflexi was the dominant microorganism when the modified biochar was added to the lower, middle, and upper parts of the reactor and mixed in the reactor, accounting for 54%, 56%, 58%, and 47%, respectively, on day 45. With the increased upward flow rate, the abundance of Bacteroidetes and Chloroflexi increased, while Proteobacteria and Firmicutes decreased. It was worth noting that the best COD removal effect was achieved when the anaerobic reactor upward flow rate was v2 = 0.6m/h and the modified biochar was added in the upper part of the reactor, during which the average COD removal rate reached 96%. In addition, mixing modified biochar throughout the reactor while increasing the upward flow rate provided the greatest stimulus for the secretion of tryptophan and aromatic proteins in the sludge extracellular polymeric substances. The results provided a certain technical reference for improving the efficiency of anaerobic digestion of kitchen waste and scientific support for the application of modified biochar to the anaerobic digestion process.