L-Homoalanine, a nonnatural amino acid that is rarely found in human and microorganisms, is used in the synthesis of various medically pivotal antiepileptic drugs and antituberculosis compounds. L-Homoalanine can be synthesized by different enzymatic approaches. In this article, the synthesis of L-homoalanine from L-methionine was explored by coimmobilization of Aspergillus flavipes L-methioninase (AfMETase) and glutamate dehydrogenase (GDH) on polyacrylamide and chitosan. Polyacrylamide coimmobilized AfMETase and GDH displayed a maximum reactivity for the synthesis of homoalanine from L-methionine. The chitosan-coimmobilized AfMETase and GDH retain about 70% of their initial activity of L-homoalanine production by the fifth catalytic reusability cycle as compared with 50% for polyacrylamide coimmobilizate. Catalytic conditions were optimized for the maximum yield of homoalanine. Homoalanine was purified by cationic and anionic chromatographs and the proton nuclear magnetic resonance (H-NMR) analysis of the lyophilized sample displayed a unique chemical structure identical to the authentic homoalanine. Using dependable dual action of AfMETase and GDH immobilized on a solid support is a novel approach for in vitro enzymatic synthesis of L-homoalanine from L-methionine, and the immobilized enzymes can be reused many times without any significant loss of their activities