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With the maturation of bio-hydrogen production technology, Nima Nasiria and others' studies showed that waste biomass such as straw can be used to produce hydrogen directly. Nowadays, due to different composition of different waste biomass, looking for the best pretreatment methods of different biomass waste on bio-hydrogen production had become a research hotspot. In this study, important factors (concentration, powder size, heating temperature, heating time, solid-liquid ratio, etc.) of four pretreatment methods (dilute acid, alkali, microwave, and enzyme) were investigated to increase the production of reduced sugars from corn straw by using orthogonal method. Orthogonal test results showed that: dilute acid was the best pretreatment method; variance analyses of experimental data showed that the weighting of different factors was: acid concentration> temperature> straw fineness> material moisture content> heating time. Among the pretreatment methods, the optimal performance was presented under the enzyme pretreatment with the value of 14.4 mg reduced sugar/100 mg, followed by the dilute acid pretreatment (11.5 mg/100 mg), the alkali pretreatment (6.14 mg/100 mg), the dilute acid pretreatment with microwave (4.29 mg/100 mg), and the alkali pretreatment with microwave (only 0.308 mg/100 mg). In addition, combined alkali and enzyme pretreatment resulted in significantly higher values of reduced sugars production from corn straw. [ABSTRACT FROM AUTHOR]