Three common pretreatments (mechanical, steam explosion and chemical) used to enhance the biodegradability of rice straw were compared on the basis of bio-hydrogen production potential while co-digesting rice straw with sludge under mesophilic (37 °C) and thermophilic (55 °C) temperatures. The results showed that the solid state NaOH pretreatment returned the highest experimental reduction of LCH (lignin, cellulose and hemi-cellulose) content and bio-hydrogen production from rice straw. The increase in incubation temperature from 37 °C to 55 °C increased the bio-hydrogen yield, and the highest experimental yield of 60.6 mL/g VSremoved was obtained under chemical pretreatment at 55 °C. The time required for maximum bio-hydrogen production was found on the basis of kinetic parameters as 36 h⁻47 h of incubation, which can be used as a hydraulic retention time for continuous bio-hydrogen production from rice straw. The optimum pH range of bio-hydrogen production was observed to be 6.7 ± 0.1⁻5.8 ± 0.1 and 7.1 ± 0.1⁻5.8 ± 0.1 under mesophilic and thermophilic conditions, respectively. The increase in temperature was found useful for controlling the volatile fatty acids (VFA) under mechanical and steam explosion pretreatments. The comparison of pretreatment methods under the same set of experimental conditions in the present study provided a baseline for future research in order to select an appropriate pretreatment method.