Abstract: To achieve efficient chitosan oligosaccharide preparation, chitosan oligosaccharide was prepared from chitosan through a pre-ultrasound-microwave assisted composite enzymatic method. The enzymatic hydrolysis process conditions were optimized using reducing sugar content as an indicator based on the response surface design method. In addition, the antioxidant capacity of chitosan oligosaccharide was examined. The results showed that the composite enzymes consisting α-amylase and chitosanase (1:1) possessed superior hydrolysis ability for chitosan than others. The optimum conditions of chitosan oligosaccharide preparation were as follows: A microwave power of 200 W, ultrasonic power of 200 W, 5 min pre-ultrasound-microwave time, 1% substrate concentration, pH5.8, temperature at 53.5 ℃, 0.24% composite enzyme additive amount, and 5.7 h enzymatic hydrolysis time. Under these conditions, the reducing sugar content reached the maximum (8.10 mg/mL). The prepared chitosan oligosaccharide contained 2~6 sugars with average molecular weight of 993.5 Da. The chitosan oligosaccharide had strong free radical scavenging activity, with the IC₅₀ of DPPH and ABTS⁺ free radicals at 0.274 and 0.127 mg/mL, respectively, as well as the ferric reducing power reached the maximum value of 24.56 mmol/L at 1.5 mg/mL. The results show that the ultrasound-microwave pretreatment combined with composite enzymatic method has a good application prospect in the preparation of chitosan oligosaccharide.