Glycerol is a readily available and inexpensive substance that is mostly generated during biofuel production processes. In order to ensure the viability of the biofuel industry, it is essential to develop complementing technologies for the resource utilization of glycerol. Ethylene glycol is a two-carbon organic chemical with multiple applications and a huge market. In this study, an artificial enzymatic cascade comprised alditol oxidase, catalase, glyoxylate/hydroxypyruvate reductase, pyruvate decarboxylase and lactaldehyde:propanediol oxidoreductase was developed for the production of ethylene glycol from glycerol. The reduced nicotinamide adenine dinucleotide (NADH) generated during the dehydrogenation of the glycerol oxidation product d-glycerate can be as the reductant to support the ethylene glycol production. Using this in vitro synthetic system with self-sufficient NADH recycling, 7.64 ± 0.15 mM ethylene glycol was produced from 10 mM glycerol in 10 h, with a high yield of 0.515 ± 0.1 g/g. The in vitro enzymatic cascade is not only a promising alternative for the generation of ethylene glycol but also a successful example of the value-added utilization of glycerol.