Biochar addition is recognized as a promising way to increase the potential of anaerobic digestion. However, few studies have investigated the interaction between biochar leaching in complex, practical anaerobic digestion systems and the negative effects that biochar leachate may have on the anaerobic digestion process. The traditional measurement methods are relatively simple, but may underestimate the complexity of dissolved organic matter in biochar leachate. In this study, the leaching behaviors of biochar and the influence of biochar leachate on anaerobic digestion were studied at the molecular level by applying ultra-performance liquid chromatography online coupled with a hybrid quadrupole orbitrap mass spectrometer (UPLC Orbitrap MS/MS) test analysis and various molecular information-based analytical methods such as van Krevelen diagrams and by conducting a series of anaerobic digestion experiments. Dissolved organic matter leaching was higher in buffered saline solution and methanol than in ultrapure water and the dissolved organic matter mainly consisted of lipids and proteins. In the lactic acid solution, a large amount of dissolved organic matter was leached from the biochar, dominated by low molecular weight dissolved organic matter such as CHO and CHON, and the lower the pH, the more was leached, suggesting that lactic acid may have an inhibitory effect on anaerobic digestion. The maximum methane production amount and microbial abundance especially key methanogenic archaea such as Methanosaeta in the sample with biochar leachate addition were lower than those for the other samples. This meant that the dissolved organic matter in the biochar leachate might inhibit the growth of microorganisms and adversely affect the acetic acid methanogenesis process. The results from this study showed that a greater number of substances with more complex structures were leached out than had been detected in previous studies. When applying biochar to anaerobic digestion in the future, it is recommended that biochar is leached first to remove toxic and detrimental substances to prevent possible inhibitory effects.