Vanillin is a well-known fragrant, flavoring compound. Previously, we established a method of coenzyme-independent vanillin production via an oxygenase from Caulobacter segnis ATCC 21756, called Cso2, that converts 4-vinylguaiacol to vanillin and formaldehyde using oxygen. In this study, we found that reactive oxygen species inhibited the catalytic activity of Cso2, and the addition of catalase increased vanillin production. Since Escherichia coli harbors catalases, we used E. coli cells expressing Cso2 to produce vanillin. Cell immobilization in calcium alginate enabled the long-term use of the E. coli cells for vanillin production. Thus, we demonstrate the possibility of using immobilized E. coli cells for both continuous and repeated batch vanillin production without any coenzymes.