The soybean milk was obtained by coldpressing from commercially available soybeans, the obtained milk was then subjected to high-pressure homogenization pretreatment assisted by different proteases (alkaline protease, papain, bromelain) to hydrolyze soybean milk and assess the effect on its structure and quality. The results regarding physical properties revealed that with the increase of homogenization pressure the solubility and hydrolysis degree of soybean milk reached 91.9% and 8.24%, respectively. Meanwhile, there was a substantial improvement in stability, negative surface charge and uniform particle size distribution. SDS-PAGE electrophoresis, infrared spectroscopy and fluorescence spectroscopy confirmed the changes in protein structure of soybean milk under different homogeneous pressures assisted enzymatic hydrolysis, showing a decreased band depth anti-nutritional protein factors. There was reduction in secondary structure and the contents of α-helix, β-rotation. The anti-nutritional factors revealed the relationship between protein structure and function. When the homogenization pressure was set at 100MPa, the inhibitory rates of the three enzymes on soybean globulin, β-concomitant soybean globulin, phytic acid, soybean lectin, trypsin inhibitor and lipoxygenase were up to 51.28%, 57.83%, 72.31%, 71.4%, 89.55% and 82.96%, respectively. The results showed that high pressure homogeneous pretreatment effectively improved the hydrolysis of protease anti-nutritional factor components. The obtained results might be fruitful for the preparation of nutritious and healthy soybean milk.