Currently, the biotechnological preparation of fragrances using natural materials attracted growing attention. Enzymatic synthesis of vanillin from isoeugenol by recombinant isoeugenol monooxygenase from Pseudomonas nitroreducens Jin1 was systematically investigated herein. With series of work on the construction of recombinant E. coli over-expressing isoeugenol monooxygenase, optimization of the culture conditions for enzyme production and reaction process for converting isoeugenol into vanillin, an increase of 22-fold in the enzyme activity (2050 U/L) was obtained, and the conversion was significantly increased at high substrate concentration with the aid of magnetic chitosan membrane for product isolation in situ. Under optimal conditions, the product concentration and space-time yield reached 252 mM and 115 g/L/d, respectively, and vanillin was obtained in 82.3% yield and > 99% purity in the gram preparative scale. The developed bioprocess showed application potential for efficient preparation of vanillin from inexpensive natural resources.