Abstract Multi‐enzymatic cascade reaction provides a new avenue for C─C coupling directly from CO 2 under mild conditions. In this study, a new pathway with four enzymes including formate dehydrogenase (PaFDH), formaldehyde dehydrogenase (BmFADH), glycolaldehyde synthase (PpGALS), and alcohol dehydrogenase (GoADH) is developed for directly converting CO 2 gas molecules to ethylene glycol (EG) in vitro. A rhodium‐based NADH regeneration electrode is constructed to continuously provide the proton and electron of this multi‐enzymatic cascade reaction. The prepared electrode can reach the Faradaic Efficiency (FE) of 82.9% at −0.6 V (vs. Ag/AgCl) and the NADH productivity of 0.737 mM h −1 . Shortening the reaction path is crucial for multi‐enzymatic cascade reactions. Here, a hydrogen‐bonded organic framework (HOF) nano‐reactor is successfully developed to immobilize four enzymes in one pot with a striking enzyme loading capacity (990 mg enzyme g −1 material). Through integrating and optimization of NADH electro‐regeneration and enzymatic catalysis in one pot, 0.15 mM EG is achieved with an average conversion rate of 7.15 × 10 −7 mmol CO 2 min −1 mg −1 enzymes in 6 h. These results shed light on electro‐driven multi‐enzymatic cascade conversion of C─C coupling from CO 2 in the nano‐reactor.