To establish high-efficiency pretreatments of cornstalk (CS) for hydrogen fermentative production, various pretreatment strategies have been investigated and contrasted in this work. Five pretreatment methods, including acid-soaking pretreatment, base-soaking pretreatment, high-temperature-assisted acid pretreatment, high-temperature-assisted base pretreatment and ultrasonic-assisted acid pretreatment (UAP), were performed on CS. The results showed that UAP significantly promoted the hydrogen production by CS compared with other pretreatments. The optimum UAP process, pretreating substrate with ultrasonication in 2.0% sulfuric acid solution for 1.5 h at the liquid-solid ratio of 20:1, obtained the maximum specific hydrogen accumulation of 142.59 mL g(-1)-CS and an average hydrogen production rate of 17.03 mL g(-1)-CS h(-1). Furthermore, the scanning electron microscope analysis of CS samples supports the hydrogen production results as well. The present work demonstrates that UAP is an efficient and practical CS pretreatment for hydrogen production from agricultural waste straws.