The low-pressure wet oxidation was used to solubilize the waste-activated sludge for recovering the carbon source, and the treated sludge was applied for the fermentative hydrogen production. The experimental results showed that, after low-pressure oxidation treatment, the concentration of soluble chemical oxygen demand (SCOD), polysaccharides, and protein present in the liquid phase was increased by 2.0, 2.2, and 102.5 times, respectively. Bio-hydrogen production was successfully achieved using solubilized sludge as the substrate. Through comparison of the hydrogen production from glucose, the treated sludge, and the mixture of the sludge and glucose, it was found that the hydrogen yield of tests using different substrates showed a positive relation with the ratio of acetic acid/butyric acid in soluble metabolites and the ratio of polysaccharides/SCOD in substrates. It can be concluded that the waste-activated sludge treated by low-pressure wet oxidation can be used as low-cost substrate for bio-hydrogen production.