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The enzymatic hydrolysis and biogas fermentation of anaerobically digested fiber from volatile fatty acids (VFAs) production with rice straw were studied. The effects of two-step pretreatment of acetic acid and aqueous ammonia on the degradation of lignocellulose, glucose concentration, xylose concentration and biogas production were explored. The results showed that two-step pretreatment of acetic acid and aqueous ammonia can further degrade the hemicellulose and lignin in anaerobically digested fiber from VFAs production, and the recovery rate of cellulose was higher, which was 77.47%~81.44%. Among them, the mass fraction of cellulose was 56.82% and the mass fraction of hemicellulose was only 7.94% for pretreatment of 4% acetic acid and 10% aqueous ammonia at 121℃. Under the above pretreatment conditions, the enzymatic saccharification efficiency was the highest, the mass concentration of glucose was 47.46g/L, and the glucose yield was 67.73%. Furthermore, the biogas production rate of anaerobically digested fiber from volatile fatty acids production pretreated by two-step pretreatment was better than those of acetic acid pretreatment alone and aqueous ammonia pretreatment alone. The highest biogas yield (414mL/g) was obtained. Two-step pretreatment effectively improved the biogas yield of anaerobically digested fiber from VFAs production. Therefore, the research proved a new biorefinery way of anaerobically digested fiber.