Our previous studies have shown that ultrasound-treated γ-aminobutyric acid (GABA)-rich coffee leaves have higher antioxidant and angiotensin I converting enzyme inhibitory activity. However, whether they have antidiabetic activity remains unknown. In this study, we aimed to investigate the inhibitory activities of coffee leaf extracts prepared with (CLE-U) or without (CLE-NU) ultrasound pre-treatment on α-amylase and α-glucosidase. Subsequently, we evaluated the binding interaction between CLE-U and both enzymes using multi-spectroscopic and in silico analyses. Ultrasound pre-treatment increased the inhibitory activities of CLE-U against α-amylase and α-glucosidase by 21.78% and 25.13%, respectively. CLE-U reversibly inhibit both enzymes, with competitive inhibition observed for α-amylase and non-competitive inhibition for α-glucosidase. The static quenching of CLE-U against both enzymes was primarily driven by hydrogen bond and van der Waals interactions. The α-helical of α-amylase and α-glucosidase was increased by 1.8% and 21.3%, respectively. Molecular docking results showed that the key differential compounds, including mangiferin, 5-caffeoylquinic acid, rutin, trigonelline, GABA, caffeine, glutamate et al. present in coffee leaves, interacted with specific amino acid residues located at the active site of α-amylase (ASP197, GLU233, and ASP300). The binding of α-glucosidase and these bioactive components involved amino acid residues, such as PHE1289, PRO1329, and GLU1397 located outside the active site. Ultrasound-treated coffee leaves are potential anti-diabetic substances, capable of preventing diabetes by inhibiting the activities of α-amylase and α-glucosidase, thus delaying starch digestion. Our study provides valuable information to elucidate the possible anti-diabetic capacity of coffee leaves through the inhibition of α-amylase and α-glucosidase activities. This article is protected by copyright. All rights reserved.