Hericium erinaceus is a well-known edible fungus rich in β-glucans, widely recognized for its immune-boosting and prebiotic properties. This study used a pulsed electric field (PEF) combined with alkaline extraction to improve β-glucan yield from H. erinaceus by-products. The treated residues were extracted with hot water or 7.5% NaOH. The results exhibited that PEF pretreatment followed by NaOH extraction gave the highest β-glucan yield (25 g/100 g) and purity (56.93%). SEM images revealed greater cell wall damage in NaOH-treated samples, while FTIR spectroscopy confirmed clear β-glycosidic linkages. The optimal conditions of PEF investigated by response surface methodology (RSM) were electric field strength 10 kV/cm, frequency 12 Hz, and mushroom/water ratio 8.44%, yielding β-glucan content of 50.14%. The extracted β-glucan demonstrated high prebiotic potential, supporting probiotic Lactobacillus spp. growth, enhancing short-chain fatty acids production, and resisting gastrointestinal digestion. Overall, this study demonstrates the broader potential of PEF-assisted alkaline extraction to support sustainable food processing, valorization of agro-industrial by-products, and the development of functional ingredients for modern food industry applications.