Fumarate　is　a　value-added　chemical　that　is　widely　used　in　food,　medicine,　material,　and　agriculture　industries.　With　the　rising　attention　to　the　demand　for　fumarate　and　sustainable　development,　many　novel　alternative　ways　that　can　replace　the　traditional　petrochemical　routes　emerged.　The　in　vitro　cell-free　multi-enzyme　catalysis　is　an　effective　method　to　produce　high　value　chemicals.　In　this　study,　a　multi-enzyme　catalytic　pathway　comprising　three　enzymes　for　fumarate　production　from　low-cost　substrates　acetate　and　glyoxylate　was　designed.　The　acetyl-CoA　synthase,　malate　synthase,　and　fumarase　from　Escherichia　coli　were　selected　and　the　coenzyme　A　achieved　recyclable.　The　enzymatic　properties　and　optimization　of　reaction　system　were　investigated,　reaching　a　fumarate　yield　of　0.34　mM　with　a　conversion　rate　of　34%　after　20　h　of　reaction.　We　proposed　and　realized　the　conversion　of　acetate　and　glyoxylate　to　fumarate　in　vitro　using　a　cell-free　multi-enzyme　catalytic　system,　thus　providing　an　alternative　approach　for　the　production　of　fumarate.　©　2023　The　Authors