The study on the rheological behavior of soybean protein isolate under different conditions is helpful to understand the formation process of fibrous structure of textured plant protein produced by extrusion. Taking soybean protein isolate as experimental material, the changes of apparent consistency of soybean protein isolate under the two modes of continuous stirring for 50min, and intermittent stirring (stirring for 10min-pause for 30min-stirring for 10min) in a biaxial kneading and mixing instrument at 80℃, moisture content of 50%, 65% and 80%, respectively. The micro rheological properties of soybean protein isolate were characterized by diffusing wave spectroscopy.The results showed that with the increase of moisture content from 50% to 80%, the longer the time required for soybean protein isolate to reach the peak apparent consistency, the greater the peak consistency. The consistency curves of 50% and 65% moisture content first increased and then decreased to a stable trend, and the consistency curve of 80% moisture content increased in stages. The decrease range of apparent consistency of 10min after reaching the peak value showed that the protein system formed by higher moisture content (65%) and continuous stirring had a smaller decrease range, which was more stable than the system formed by 50% moisture content and intermittent stirring. The diffusion wave spectroscopy test showed that when the moisture content was low (50%), the protein tended to form a structure with large grid(55nm),poor continuity and low storage modulus. When the water content is increased to 65%-80%, or the water content was high (65%) combined with continuous stirring, the grid was small(5-11nm) and the storage modulus was high. It was concluded that the moisture content had a great influence on the cross-linking behavior of protein in kneading, and reducing the moisture weakened the ability of protein to form network structure. Increasing water content could promote protein aggregation and reduce the grid size. Continuous mechanical stirring could also form protein continuous structures with small grids.