The invention discloses a high-protein straw feed, which comprises crop straw powder, actinomycetes, nocardia, lactobacillus acidophilusis and bacillus subtilis, the weight ratio of the strains is 0.8-1.2:1.6-2.4:0.8-1.2:1.8-1.2, and the weight ratio of the total strain amount to the crop straw powder is 1:1,000-1,200. The preparation method comprises the following steps of: respectively screening, separating, purifying, rejuvenating and culturing various strains, transplanting the strains into an industrial fermentation tank for fermentation, mixing in a ratio, and adsorbing by using an adsorptive support; and inoculating the obtained composite viable bacteria powder into crop straw powder or a mixture of the crop straw powder, staking and fermenting at the temperature of between 60 and 70 DEG C, uniformly turning once every two days, and obtaining the high-protein straw feed after 30 to 35 days. The high-protein straw feed has the advantages of improving protein content, optimizing protein structure, improving the absorption and nutrient conversion rate of the straw feed, and improving the immunity of livestock.