To make the previously developed biosynthesis of l-2-aminobutyric acid (l-ABA) more suitable for the industrial-scale production. A recyclable biotransformation system was developed based on immobilized enzyme technology. The conversion yield of l-threonine (at 90 g l(-1)) reached 99.9 % and the theoretical yield of L-ABA reached more than 90 % using the optimized biotransformation system by the individual immobilization of threonine deaminase and the co-immobilization of l leucine dehydrogenase and formate dehydrogenase. 90 g l-threonine l(-1) was converted to 73.9 g l-ABA l(-1) > 95 % theoretical yield, within 120-145 min in 30 batch transformation experiments. The recyclable biotransformation system is promising to fulfill industrial requirements for l-ABA production.