A new type of three-dimensional (3D) serpentine microchannel reactor was fabricated by thermally bonding stacked polyimide films to greatly improve the mixing efficiency by transversal vortices at low Reynolds number (Re). The effects of Re, flow ratio R, and channel width on the mixing performance were investigated using the Villermaux-Dushman method, in which the segregation index X-s was utilized to quantify the mixing efficiency. The results showed that X-s decreased with increasing Re, decreasing R, and reducing channel width. Then, a computational fluid dynamics model was employed to analyze the flow characteristics. The vortices that varied periodically along the 3D serpentine microchannel significantly improved the mixing efficiency. Furthermore, the application of the 3D serpentine microchannel reactor in the biohydration of acrylonitrile demonstrated that the new design is very promising for fast reactions that are limited by mass transfer resistance.