The decrease of cellulase activity and unproductive adsorption of lignin are important obstructive factors for inefficient enzymatic hydrolysis. This paper applied five different kinds of biosurfactants including rhamnolipid, sophorolipid, chitin, tea saponin, and sodium lignosulfonate in the enzymatic hydrolysis process of alkali-pretreated reed straw (RS) to enhance the saccharification efficiency. When 8 g/L sophorolipid is added, the efficiency of enzymatic hydrolysis is 91.68 %, which is 30.65 % higher than that without using any biosurfactant. The efficiency of enzymatic hydrolysis can be further increased to 99.56 % when 7.5 g/L sophorolipid and 1.5 g/L tea saponin are added together. This is because the sophorolipid, rhamnolipid, and chitin can synergistically hamper the enzymatic inactivation during enzymatic hydrolysis, while tea saponin and sodium lignosulfonate can inhibit the non-productive adsorption of lignin. This work proposed a very effective method to improve the efficiency of enzymatic hydrolysis and reduce the dosage of the enzyme by adding biosurfactants.