The utilization of biochar-immobilized microorganisms is an effective method for eliminating phenol from water. However, the main challenge to the practical implementation of this method is the high susceptibility of bacteria to environmental factors. In this study, biochar was used to reduce microbial susceptibility to high concentrations of phenol. Demineralized biochar obtained at high temperatures (600/800/1000 C) was used to immobilize phenol-degrading bacteria (PDB) and enhance phenol removal. Biodegradation was inhibited by high concentrations of phenol (>1300 mg/L). In contrast, when microorganisms were combined with biochar, up to 82.4% phenol removal occurred, surpassing the efficacy of phenol degradation by a single microorganism (42%). Characterization techniques such as SEM, XRD, and XPS were used to analyze the properties and immobilization effect of biochar. The addition of biochar altered the dominant species of PDB to include Serratia, Ralstonia and Enterobacter. Response surface analysis indicated that both biochar pyrolysis temperature and experimental temperature significantly influenced the phenol removal rate. Thus, this paper provides methodological guidance for the application of biochar-immobilized microorganisms for phenol wastewater treatment.