Anaerobically activated promoters in Escherichia coli play crucial roles in transcriptional regulation during cellular responses to decreased oxygen concentrations and serve as essential tools for implementing dynamic regulation in metabolic engineering. These promoters exhibit transcriptional activity only under low-oxygen or anaerobic conditions. To discover novel anaerobically activated promoters, this study selected 11 native promoters from E. coli databases and characterized their activities using flow cytometry. Subsequently, we optimized the key elements of these promoters and re-evaluated their activities to investigate the impact of functional elements on promoter performance. Furthermore, we verified the regulatory mechanisms of these promoters by knocking out host regulatory genes. Finally, we characterized the promoters' responsiveness to aerobic-anaerobic transitions by rapidly switching cultivation environments during host growth. This study identified several novel anaerobically activated promoters and comprehensively characterized their performance and features from multiple aspects. The identified promoters provide new tools for oxygen-limited or anaerobic production in metabolic engineering, while the findings from promoter element optimization offer valuable references for the design of anaerobically activated promoters.