In order to improve the quality of highland barley flour and the taste of highland barley biscuit which is rough and difficult to swallow, the semi-solid fermentation of highland barley flour with transglutaminase was performed, and the quality of highland barley flour and the taste of highland barley biscuit were investigated. The optimum conditions were selected via evaluating swelling index of glutenin, water retention and total sugar content before and after fermentation. Sensory scores of highland barley biscuits before and after fermentation were compared. The results showed that with the conditions of fermentation temperature 25℃, enzymatic time 5h, and enzyme mass fraction 0.3%, the swelling index of glutein, water retention rate, and total sugar content were 3.36,60.70%, and 7.19mg/mL, respectively. In comparison with biscuit of highland barley, the biscuit of fermented highland barley was easy to form gluten during the making process and had better taste and pleasant aroma.