This study investigated the effect of Lactobacillus plantarum DM616 on dough fermentation for making Chinese steamed bread. Dough fermentation was carried out via inoculation with Saccharomyces cerevisiae as monoculture, or in combination with L. plantarum DM616 as co-culture. The presence of L. plantarum DM616 caused faster depletion of reducing sugars and had little effect on the maximum specific growth rate (μmax) and final cfu/g sourdough of S. cerevisiae. In co-culture, the pH of the dough varied from 3.2 to 3.5 and the total titratable acids developed to 9.2 after 24 h of fermentation. L. plantarum DM616 did not significantly suppressed the leavening capacity of the S. cerevisiae. Scanning electron microscopy results showed that the microstructure of the co-culture fermented dough presented a more continuous gluten network structure, as compared with the monoculture sample. A positive effect of L. plantarum DM616 on the hardness and whiteness of steamed bread was observed after 16 h of fermentation. Practical Applications Lactobacillus plantarum DM616 isolated from traditional leavening agents can be used for dough fermentation for making Chinese steamed bread. Application of DM616 could improve the hardness and whiteness properties of steamed bread. The research provided useful information for the suitability of co-culture in sourdough fermentation for production of steamed bread.