Abstract: Abstract: In the process of anaerobic fermentation, the material stratification and the parameters of the stratified fermentation fluid, such as viscosity and density, are the key foundation of the stirring device design. In this paper, the relay methods were adopted to investigate the variation of the basic properties of stratified anaerobic fermentation fluid in the process of cow dung anaerobic fermentation at mesophilic temperature. The results demonstrated that the densities of the fermentation fluid increased from top to bottom in the reactor, and increased along with the increase of substrate concentration. In the process, the maximum density of each stratified fluid appeared on the 4th day; when Total solid were 4%, 6% and 8%, the maximum densities were 1.02, 1.02 and 1.03 g/cm3 in the top layer, 1, 1.03 and 1.03 g/cm3 in the middle layer, and 1.02, 1.07 and 1.07 g/cm3 in the bottom layer, respectively. The viscosities of fermentation fluid in the top and middle layer of the reactor increased in primary stage, then got decreasing and tended to be stable at last under the influence of the liquid stratification. The maximum viscosity of the liquid in the bottom layer was the initial viscosity, and the initial substrate concentration affected the viscosity change significantly. When TS was 4%, the viscosities of the fluid in the top and middle layer reached the maximum on the 7th and 4th day, respectively, and the maximum viscosities were 11.5 and 14.7 mPa?s, respectively; the maximum viscosity of bottom layer was 107 mPa?s. When TS was 6%, the viscosities of the fluid in the top and middle layer reached the maximum on the 4th day, and the maximum viscosities were 25.5 and 15.5 mPa?s, respectively; the maximum viscosity of bottom layer was 135.5 mPa?s. When TS was 8%, the viscosities of the fluid in top and middle layer reached the maximum on the 4th day, and the maximum viscosities were 63.5 and 95.5 mPa?s, respectively; the maximum viscosity of bottom layer was 185.5 mPa?s. Daily gas yield increased and the gas peak appeared earlier with the increase of the substrate concentration. The cumulative gas yield with the TS of 8% was 21.4% and 8.71% higher than those with the TS of 6% and 4%, respectively, while the increase of methane content of the biogas was almost the same, and basically stable around on the 10th day.