Microbial fuel cell (MFC) is applied to produce electricity using dairy manure as a fuel. Since the way MFC utilizes manure as a fuel and the long-term operation stability of manure-MFC remains unclear, this study examined the evolution of dissolved organic matter (DOM) in anodic chamber and power generation by MFC in a 171days test. The tested MFC can produce electricity over the entire testing period by single feed of manure, with stable power output and total chemical oxygen demand (TCOD) removal rate in the period of day 30–140. The hydrophobic acid (HPO-A) and hydrophilic (HPI) fractions of manure were the principal components of anolyte DOM, with the concentrations of both being reduced over MFC operation. The degradable organic matters were converted to compounds with high aromaticity.