The present study evaluated the optimal parameters for Psidium guajava leaf extraction by ultrasound to obtain maximal anti-hyperglycemic activities in the extract. The response surface methodology was employed for empirical model building. The maximum inhibition of the two starch-digesting enzymes α-amylase and α-glucosidase was determined. The optimized extraction parameters of solvent-to-solid ratio, extraction time, and extraction temperature for obtaining the maximum inhibitory activity for 50 µg/mL extract were determined to be the following: 12.07 (v/w), 5.22 min, and 59.77℃ for α-amylase (47.23% ± 0.76%) and 12 (v/w), 5.22 min, and 59.94℃ for α-glucosidase (59.42% ± 0.65%), respectively. The F values for the lack-of-fit were not significant ( p > 0.05) for both second-order models, indicating them to be appropriate for describing the response surface. In conclusion, our study presents the optimal conditions for ultrasound-assisted extraction of guava leaf as a potential substance for use in diabetes treatment.