Temperature distribution has a great influence on the efficiency of gas production for anaerobic fermentation system. Fluent is one of the most advanced software for fluid simulation. It is suitable to apply it in simulation analysis of the anaerobic reactor without stirring. Two kinds of raw materials which were water and digested slurry with TS of 12.4% were applied in two 10L anaerobic reactors to conduct anaerobic fermentation experiments. Totally 16 temperature sensors were installed at each anaerobic reactor in order to detect temperature continuously. The temperature figures indicated that two types of materials were definitely different. Therefore, applying the Fluent heat transfer simulation to digested slurry anaerobic fermentation system cannot only utilize water as physical parameter. However, the error was only 4.2% between experiment with raw material of digested slurry with TS of 12.4% and Fluent simulation, when water and Boussinesq hypothesis were simultaneously defined as physical parameters. It was also explored that setting up a virtual wall in Fluent can dramatically simplify the model calculation time, and it had the same effect with defining real wall model in Gambit. In addition, it was found that realizable k—ε model was the most suited to the anaerobic fermentation system of digested slurry with high TS. The aims of the research were to explore suitable Fluent setting and optimization method for anaerobic fermentation and provide guidance for further work. Optimizing model and improving efficiency in unsteady-state numerical analysis will be the main body of the further work. 