A method for γ-aminobutyric acid (GABA) production was studied using highland barley bran combined with adding sodium glutamate and pyridoxal 5'-phosphate (PLP). Based on the influences of different varieties on GABA bioconversion and the single test of factors including solution pH， initial whole flour concentration， reaction temperature and time， initial concentrations of sodium glutamate and PLP， the optimal reaction conditions of GABA production through the orthogonal experiment were developed. This research further showed that a strong influence of peeling level of barley bran on GABA enrichment. A safe and highly efficient preparing conditions without any buffer was determined as follows: bran peeling rate 4.23%-6.43%， barley bran concentration 140∶1 (g/L)， reaction time 12 h， reaction temperature 35 ℃， 8 mmol/L and 18 μmol/L supplemental level of sodium glutamate and PLP， respectively. The optimal reactions produced 19.57 mmol/L GABA from 8 mmol/L sodium glutamate， with a GABA conversion rate up to 97%.