The invention discloses an active Ganoderma lucidum polysaccharide peptide reference substance, and a preparation method and application of the reference substance, and provides an active Ganoderma lucidum polysaccharide peptide reference substance for measuring and evaluating the quality of Ganoderma lucidum and Ganoderma lucidum products. The reference substance can correct the errors in polysaccharide measurement generated using glucose as the reference substance after addition of starch as an adjuvant during the deep processing process of Ganoderma lucidum. The preparation method uses Ganoderma lucidum as a raw material and comprises the steps of soaking in water, boiling, collecting an extractive solution, concentrating, centrifuging, dialyzing, entrapping polysaccharide protein GL-PP with an active macromolecular site, further carrying out ethanol gradient separation of the GL-PP, centrifuging, and collecting precipitates; and separately carrying out column separation, and selecting active polysaccharide peptide GL-PPS as the reference substance for detecting polysaccharide peptides, wherein the reference substance can be used for accurate measurement of the content of trace active polysaccharide peptides in a UV (ultraviolet) instrument. The pure product of the reference substance disclosed by the invention can be used for detecting the quality of Ganoderma lucidum products, and can eliminate the interference of starch and other adjuvants in the Ganoderma lucidum products, so as to improve the quality standards of Ganoderma lucidum products.