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The enzymatic hydrolysis and biogas fermentation of rice straw were studied. The effects of combined ozone and soaking aqueous ammonia pretreatment on the degradation of lignocellulose, monosugars concentration, reducing sugars concentration and biogas production were explored. The results showed that combined ozone and aqueous ammonia pretreatment further increased the delignification ability, improved the saccharification efficiency of rice straw and biogas yield, and enhanced the fiber degradation efficiency. During enzymatic hydrolysis, the glucose concentration was increased first and then decreased with the increase of ozone dosage. When the ozone dosage was 0.75g/g, the glucose concentration was the highest, as 36.92g/L. The longer ammonia pretreatment time was, the higher the reducing sugar concentration was, and the ammonia concentration for 6h ammonia pretreatment was 60.51g/L. Therefore, the suitable combined pretreatment conditions for enzymatic hydrolysis was 0.75g/g ozone dosage and 6h of soaking aqueous ammonia. For biogas fermentation, a high methane yield of 165.39mL/g was obtained with 1.0g/g of ozone dosage and 9h of soaking aqueous ammonia time, which effectively increased the gas production efficiency of rice straw and utilization efficiency of lignocellulosic materials.