Abstract: Multi-enzyme-assisted laminarin extraction parameters were optimized, and then the prepared laminarin was assessed for inhibition of α-glucosidase activity. The relative proportions of preparative enzymes were investigated to maximize laminarin yield using an orthogonal test design. The effect of enzymatic hydrolysis time, pH, ratio of solid to liquid and temperature on laminarin yield were determined using response surface methodology (RSM). Finally, laminarin inhibition of α-glucosidase activity was assessed using an in vitro enzyme inhibition test. Results revealed that the optimal laminarin yield was obtained using a ratio by mass of 9:10:5.5 of cellulase, pectinase and papain, respectively, and an enzymatic hydrolysis time, temperature, pH and ratio of liquid to material ratio of 1.8 h, 49.4 °C, 6.1 and 59:1 mL/g, respectively. The predicted yield of laminarin under optimal conditions was 18.183%, which resembled the actual laminarin yield of 18.19%±1.04%. Its neutral sugar, acid sugar, protein and sulphate contents were 52.72%, 11.76%, 2.66% and 19.49%, respectively. As laminarin concentration increased from 1 mg/mL to 5 mg/mL, its α-glucosidase inhibition activity increased, with highest inhibition of 79.04%±3.17% and IC₅₀ value of 1.443 mg/mL. This multi-enzyme-assisted extraction procedure generated a high yield of laminarin that exerted clearly demonstrable inhibition of α-glucosidase activity.