The invention provides a preparation method of high-content Ganoderma lucidum β-glucan and an efficient and rapid detection method thereof. The preparation method comprises ethanol-trypsin pretreatment of Ganoderma lucidum fruiting body in combination with ultrasonic-assisted alkaline hydrothermal extraction of β-glucan from Ganoderma lucidum, and purifying β-glucan by DEAE Sephadex A-25 Sephadex column chromatography using NAOH solution as a mobile phase, to obtain high purity (≥90%) of Ganoderma lucidum β-glucan. The detection method comprises placing the extracted β-glucan in an autoclave and performing hydrolysis with β-(1,3)-exo-glucanase and β-glucosidase, and detecting and analyzing the content of the prepared β-glucan by high pressure-acid enzymatic hydrolysis-HPLC method, using glucose as a standard. The preparation method of β-glucan has high extraction ratio. The efficient and rapid detection method has high sensitivity, high accuracy and good repeatability, and thus has a wide application prospect.