Acorn nut flour (ANF) is universally considered as a feasible and alternative non-food starchy feedstock for fermentation industries, with an annual production of over 2 million tons in China, while it contains around 7.17% tannin content with potential effective inhibition to enzyme proteins involved in the common amylase hydrolysis of starch to fermentable glucose. Compared with corn and potato starch, the obvious damping and inhibitory rate was experimentally detected with at least 35% decline during the enzymatic hydrolysis of ANF. This decline was completely resolved by the extraction and removal of tannin from ANF, or by improving α-amylase loading; meanwhile, the return and addition of extracted tannin markedly weakened α-amylase liquefaction of varied starchy feedstocks. These data provided a solid evidence for acorn nut tannin bio-inhibition to α-amylase hydrolysis of ANF. Bovine serum albumin (BSA) and milk protein successfully substituted α-amylase as sacrifice-proteins that resolved effectively the tannin bio-barrier to α-amylase hydrolysis of ANF, and improved the enzymatic hydrolysis and glucose yield to over 86%. Finally, the common and cost-effective milk protein substitute was recommended, providing the technical guidance for new food-substitute bioresources and acorn-based industry.