Objective: This study aimed to construct an enzymolysis and membrane separation method to improve the production efficiency of low molecular hyaluronic acid (HA). Methods: A continuous enzymolysis and membrane separation coupling reaction system was designed, which consisted of an enzyme biological reaction tank and a flat polyethersulfone ultrafiltration membrane separation module. By evaluating membrane flux, membrane contamination, hyaluronic acid concentration, and yield, the effects of various factors on the separation of low molecular HA in the enzyme-membrane coupling system were systematically investigated. The process parameters of the enzyme-membrane coupling reaction were further optimized through orthogonal experiments. Results: The optimal process parameters for the preparation of low molecular hyaluronic acid by enzyme-membrane coupling reaction system were a stirring speed of 200 r/min, transmembrane pressure of 0.15 mPa, enzymolysis time of 4.0 h, and enzyme dosage of 5 g/100 g. In this method, two low molecular hyaluronic acids with different molecular weights (M_r) can be quickly prepared and separated, the low molecular weight hyaluronic acid (LMW-HA) with Mw between 10 000~50 000 and the HA oligosaccharides (O-HA) with M_r ＜3 000 can be prepared rapidly by one-step approach. Both of these different molecular weights hyaluronic acid could scavenge DPPH and hydroxyl free radicals. Conclusion: The enzymolysis and membrane separation coupling method provides an efficient method for simultaneous and continuous production of LMW-HA and O-HA with different molecular weights and certain antioxidant properties.