Protein is a potential resource for fermentation, which is one of the rate limiting factors of fermentation. This study investigated some of the regularities of protein on dark fermentation for acid production, whey (82% protein) liquid was used to study the effect of varying headspace pressure (HP; 0.02-0.10 MPa), protein concentration (6.25-18.75 g/L) and hydraulic retention time (HRT; 24-48 h) on VFA production. The main fermentation product is acetate and butyrate. Low HP, low substrate concentration and short HRT were favorable for the generation of acid. When the protein concentration was 12.5 g/L and HRT was 24 h, the VFA concentration was up to 8924.190 mg/L at 0.02 MPa, compared to a 61.55% increase under 0.10 MPa. While the HRT was 48 h or the protein concentration was 18.75 g/L, VFA concentration was low and no more n-butyric acid or valeric acid. After variance analysis, we observed a significant effect of the three factors on acid.