Display omitted •Cellulose-rich grass clipping showed great potentials for biofuel production.•Ultrasound-Ca(OH)2 was the most effective pretreatment method.•Pretreatment removed lignin and hemicellulose, and increased crystallinity index.•Reducing sugar yield of grass clipping reached 414mg/g after pretreatment. Grass clipping, a cellulose-rich raw material, has great potential to produce biofuels, but must be firstly hydrolyzed to liberate fermentable sugars. In this study, grass clipping was pretreated with ultrasound (US), Ca(OH)2, NaOH, US-Ca(OH)2 and US-NaOH at relatively low temperature to enhance its enzymatic hydrolysis. The solubilization of hemicellulose and lignin, and crystallinity index of cellulose increased after US-alkaline pretreatment, leading to a significant increase of enzyme accessibility to cellulose. Compared with another four pretreatments, US-Ca(OH)2 pretreatment of grass clipping showed the best improvement for reducing sugar yield. X-ray diffraction (XRD) determination and scanning electron microscope (SEM) observation showed that the crystallinity index of grass clipping increased and the grass clipping surface suffered from serious erosion after US-Ca(OH)2 pretreatment. Then, the operating conditions of US-Ca(OH)2 pretreatment and enzymatic hydrolysis were systematically optimized, and the suitable operating conditions were as follows: US power density of 0.65W/ml, US pretreatment time of 30min, Ca(OH)2 concentration of 0.75%, pretreatment temperature of 75°C, enzyme loading of 125FPU/g, and hydrolysis time of 72h. The reducing sugar yield of grass clipping pretreated by US-Ca(OH)2 reached 414mg/g, increasing by 3.5 times compared with that of raw grass clipping. The US-Ca(OH)2 pretreatment of grass clipping at low temperature significantly enhanced the potential of grass clipping as a promising raw material to produce biofuels.