The beneficial biological effects of Reishi mushroom, Ganoderma lucidum, are primarily due to its polysaccharides containing β-glucans. However, the rigid chemical structure of fruiting bodies in G. lucidum makes the extraction of polysaccharides more difficult compared to other edible mushrooms. Although there are a number of studies reporting various methods for extracting β-glucans from G. lucidum fruiting bodies, data on the evaluation of the effect of method of extraction on the chemical profile of β-glucans in terms of the content and the efficiency of extraction are lacking. The present study sought to evaluate 15 different basic chemical and analytical techniques, including ten conventional methods, three optimized conventional methods, and two advanced methods (enzymatic and supercritical CO2 methods) for extracting polysaccharides from G. lucidum fruiting bodies. The use of the conventional methods resulted in low amounts of glucans and extraction efficiency. Among the optimized conventional methods, the alkaline extraction method produced a high amount of β-glucan (9.30% dw) and a high extraction efficiency of about 11% dw (p ≤ 0.05). The use of β-glucanase led to the highest total glucan (16.3% dw) and β-glucan (14.73% dw) contents (p ≤ 0.05), with 10.98% dw extraction efficiency. The highest extraction efficiency (14.12% dw) was also obtained from the supercritical CO2 method (p ≤ 0.05) with 11.31% dw β-glucan. In conclusion, the most effective methods for extracting β-glucans from G. lucidum fruiting bodies include supercritical CO2 extraction, enzymatic, and alkaline methods. These findings may provide tools for strategic decision-making regarding the choice of extraction method.