In order to improve the efficiency of anaerobic digestion for biogas production and achieve the resource recovery and decrement of antibiotic bacterial residue, ultrasonic/alkaline pretreatment is used to treat penicillin bacterial residues (PBR), the influence of pH value, ultrasonic sound energy density, moisture content and reaction time on pretreatment effect are investigated, and the biodegradability of PBR is evaluated by biochemical methane potential (BMP) test. The orthogonal experiment result shows that ultrasonic/alkaline pretreatment can enhance the residue cell wall broken effect, and promote intracellular organic matter dissolution. The optimum pretreatment conditions are pH of 10, ultrasonic sound energy density of 20 W/mL, moisture content of 97%, and pretreatment time of 5 min. In this case, the highest COD dissolution rate can reach 84.69%, which is 2.08 times higher than that by the ultrasonic pretreatment only. The BMP test results show that the impact of the pretreatment factors is as follows: moisture content > ultrasonic sound energy density > reaction time > pH value. The optimum pretreatment conditions according to methane productivity are pH of 9, ultrasonic sound energy density of 0.5 W/mL, moisture content of 96%, and reaction time of 30 min. In this case, the methane yield rate can reached 335 mL/g, which is 2.2 times higher than that from the untreated bacteria residue.