In this study, the recombinant ectoine-producing Escherichia coli ET01 was constructed by introducing the ectABC operon from Halomonas venusta ZH. To further improve ectoine production, the regulation of the fermentation process was systematically investigated. First, the effects of the initial glucose concentrations and glucose feeding mode on ectoine production were analyzed. Using a combination of pH-feedback feeding and glucose-controlled feeding, the ectoine titer reached 25.5 g/L, representing an 8.8-fold increase over standard batch culture. Then, the effects of dissolved oxygen (DO) levels (50, 40, 30, or 20%) on ectoine production were studied, and a DO control strategy was developed based on the fermentation kinetics. When the final optimized two-stage fermentation strategy was used, the ectoine titer reached 47.8 g/L, which was the highest level of ectoine produced by E. coli fermentation. The fermentation regulation strategy developed in this study might be useful for scaling up the commercial production of ectoine.