In this study, zein-based gluten-free model dough (Z dough) was prepared with zein, corn starch, and wormwood gum. Based on the flour quality test, the kneading torque, water composition and protein distribution of the zein simulated dough with different lactic acid content were compared when finish dough (torque up to 1.1 Nm; set as ZS dough) and kneading for 12 minutes (set as ZL dough) conditions. The optimal amount of lactic acid added to kneadable dough was explored, and the physical and chemical properties of zein in dough with different lactic acid content were discussed under the action of kneading for a certain period of time, which could and provide reference for the preparation of related cereal products. Studies have shown that adding a small amount of lactic acid could promote zein to build a protein network in the dough, but long-term kneading would lead to decreasing the strength of the acidified dough, destroying the continuity of the zein network in the dough, reducing the level of zein wrapping on starch granules and weak bonding in the dough. The proportion of water decreased and the proportion of free water increased. The surface hydrophobicity increased and the relative molecular weight decreased, when the deamidation reaction of zein occurred. The analysis showed that the dough gradually weakened after a long time of kneading, because the kneading effect would expand the effect of lactic acid on zein in the dough, which would increase the hydrophobicity of the protein surface and fragment the protein, and lead to a decrease in the coherence of the zein network.