As a vital spice, vanillin is widely used in diverse industries. Natural vanillin cannot meet the demand of market due to many restrictions. Therefore, chemical synthesis of vanillin is the main source for market. With the continuous depletion of natural resources and the improvement of people’s awareness of environmental protection in recent years, the biological production of vanillin by microbial transformation using suitable substrates has gradually become a research hotspot. This article reviewed the related research progresses on vanillin production by bacteria and fungi with eugenol, isoeugenol and ferulic acid as substrates and also presented the investigation of the metabolic pathways of vanillin produced by eugenol, isoeugenol and ferulic acid as well as the application of biotechnology in this field. The biosynthesis of vanillin heralds vast potentials for development and considerable market prospects.