Waste activated sludge is a valuable resource containing multiple nutrients, but is currently treated and disposed of as an important source of pollution. In this work, waste activated sludge after ultrasound pretreatment was reused as multiple nutrients for biofuel production. The nutrients trapped in sludge floc were transferred into liquid medium by ultrasonic disintegration during first 30 min, while further increase of pretreatment time only resulted in slight increase of nutrients release. Hydrogen production by Ethanoligenens harbinense B49 from glucose significantly increased with the concentration of ultrasonic sludge, and reached maximum yield of 1.97 mol H2/mol glucose at sludge concentration of 7.75 g volatile suspended solids/l. Without addition of any other chemicals, waste molasses rich in carbohydrate was efficiently turned into hydrogen with yield of 189.34 ml H2/g total sugar by E. harbinense B49 using ultrasonic sludge as nutrients. The results also showed that hydrogen production using pretreated sludge as multiple nutrients was higher than those using standard nutrients. Acetic acid produced by E. harbinense B49 together with the residual nutrients in the liquid medium were further converted into hydrogen (271.36 ml H2/g total sugar) by Rhodopseudomonas faecalis RLD-53 through photo fermentation, while ethanol was the sole end product with yield of 220.26 mg/g total sugar. Thus, pretreated sludge was an efficient nutrients source for biofuel production, which could replace the standard nutrients. This research provided a novel strategy to achieve environmental friendly sludge disposal and simultaneous efficient biofuel recovery from organic waste.