Display omitted •Ultrasonic conditions significantly affect extraction efficiency.•Guava leaf polysaccharides (GLP) has good antioxidant activity.•GLP exhibits remarkable hypoglycemic activity in vitro. Plant polysaccharides possess multiple physiological activities. Box-Behnken design (BBD) was employed to optimize the ultrasound-assisted extraction of polysaccharides from guava leaves (GLP). It was found that ultrasonic time, temperature and power has a significant effect on the extraction efficiency. The yield of GLP was 1.00 ± 0.04% under the optimal condition of extraction time of 20 min, temperature of 62 ℃ and ultrasound power of 404 W. The DPPH and ABTS+ radical scavenging rate of GLP was 56.38% and 51.73% at 100 μg/mL, respectively. The rate of inhibition of α-amylase by GLP was only 14.06% at 1 mg/mL; however, its α-glucosidase inhibition rate reached 99.54% at 100 μg/mL. These results suggested that the ultrasound-assisted extraction for GLP is effective and environmentally friendly and GLP has wide application potentials as natural antioxidants and α-glucosidase inhibitors.