Vanillin is a popular flavoring compound and an important food additive. Owing to the consumer preference for inexpensive natural aroma flavors, vanillin production through a biotechnological pathway has become of great interest and commercial value in recent years. In this study, an enzymatic synthetic system for vanillin using a coenzyme-independent decarboxylase (FDC) and oxygenase (CSO2) cascade was reconstituted and optimized. This system produces a slightly higher production yield (40.20%) than the largest yield reported for immobilized FDC and CSO2 (35.00%) with ferulic acid as a substrate. It was previously reported that the low catalytic activity and thermal instability of CSO2 restrict the overall productivity of vanillin. In present study, site-directed mutagenesis was applied to rate-limiting oxygenase CSO2 to generate positive mutants. The production yields of mutants A49P (58.44%) and Q390A (65.29%) were 1.45- and 1.62-fold that of CSO2 wild type, respectively. The potential mechanism for enhanced vanillin production using A49P involved increased thermostability and catalytic efficiency, while that using Q390A was probably associated with a better thermostable performance and increased catalytic efficiency resulting from a larger entrance channel.