Taking refined flour, matsutake powder, and highland barley powder as main raw materials, this experiment studied the optimal formula of matsutake highland barley biscuit. Besides, single factor experiment was carried out for the amount of highland barley powder, white granulated sugar, and shortening. Then, the response surface optimization analysis was made on crispness and sensory score of the biscuit. The experiment indicates that taking the refined flour as the base 100 g (100%), the formula of 20% highland barley powder, 25% white granulated sugar, and 26% shortening can bake crisp biscuit with complete shape, pure flavor and high quality.