Among the human milk oligosaccharides (HMOs), one of the most abundant oligosaccharides and has great benefits for human health is 3 '-sialyllactose (3 '-SL). Given its important physiological functions and the lack of cost-effective production processes, we constructed an in vitro multi-enzymatic cofactor recycling system for the biosynthesis of 3 '-SL from a low-cost substrate. First, we constructed the biosynthetic pathway and increased the solubility of cytidine monophosphate kinase (CMK) with chaperones. We subsequently identified that beta-galactosidase (lacZ) affects the yield of 3 '-SL, and hence with thelacZgene knocked out, a 3.3-fold increase in the production of 3 '-SL was observed. Further, temperature, pH, polyphosphate concentration, and concentration of divalent metal ions for 3 '-SL production were optimized. Finally, an efficient biotransformation system was established under the optimized conditions. The maximum production of 3 '-SL reached 38.7 mM, and a molar yield of 97.1% from N-acetylneuraminic acid (NeuAc, sialic acid, SA) was obtained. The results demonstrate that the multi-enzymatic cascade biosynthetic pathway with cofactor regeneration holds promise as an industrial strategy for producing 3 '-SL.