The alkaline hydrogen peroxide (AHP) pretreatment of cladodes of cactus (Opuntia ficus-indica) for biogas production was evaluated based on the delignification of cladodes of cactus. The effects of alkaline hydrogen peroxide concentration (30% w/w solution) and the pretreatment time (3, 6, 9, and 12 h) were evaluated at pH 11.5, temperature of 30 °C, and 180 rpm for removal of lignin. A batch of anaerobic digestion experiments were conducted at mesophilic temperature conditions (37 ± 1 °C) with the pretreated biomass. The feed stock (cladodes of cactus) used in this study contained 12.51 ± 1.25 cellulose, 16.34 ± 2.93% hemicellulose, and 10.45 ± 2.31% lignin, and the balance were (carbohydrate, protein, lipid, and ash). After AHP pretreatment, the lignocellulosic content of the feed stock was changed to 12.50 ± 1.84%, 13.63 ± 3.23%, and 7.49 ± 3.05% for cellulose, hemicellulose, and lignin respectively. The AHP pretreatment of cladodes of cactus highly affected the lignin structure relative to cellulose and hemicellulose. The alkaline hydrogen peroxide pretreatment resulted in a higher amount of biogas produced from 877.9 ± 15.12 ml biogas/g VS to 1613.5 ± 10.76 ml biogas/g VS which is an 83.4% increment and decreased after 9 h treatment to 1398.8 ± 17.8 ml biogas/g VS. In addition, the measured methane yields range from 302.48 ± 0.33 to 602.65 ± 3.24 ml CH4/g VS. The results showed that alkaline hydrogen peroxide pretreatment of cladodes of cactus is an effective strategy for enhance biogas yield.