Fermentative hydrogen production was performed using waste-activated sludge pretreated by a low-frequency ultrasound. The experimental results showed that a low-frequency ultrasound can effectively solubilize the waste-activated sludge and release the organic matter for hydrogen production. The ratio of soluble chemical oxygen demand (SCOD)/total chemical oxygen demand (TCOD) achieved 20.2% after ultrasound pretreatment, and the increased SCOD was mainly comprised of dissolved polysaccharides. A hydrogen yield of 68.9 mL/g of SCOD was obtained when ultrasound-treated sludge was used as the substrate, and the linear relationship between the hydrogen yield and polysaccharide/SCOD ratio of the substrate was observed. Hydrogen production from ultrasound-treated sludge followed acetate-type fermentation.