Biochemical and pharmacological research has demonstrated that Lingzhi or Reishi medicinal mushroom Ganoderma lucidum polysaccharides (GLPS) have significant anticancer, antitumor, and antioxidant activities. To investigate the effect of injecting GLPS into hosts for clinical studies, aqueous polysaccharide extracts from G. lucidum fruit bodies were purified by deproteinization using the Sevage method, anion-exchange chromatography elution (cellulose DEAE-52 chromatography), dialysis, ethanol precipitation, and active carbon and millipore membrane filtration techniques. The purified GLPS were used for injection in mice. Polysaccharide indexes, protein, tannin, heavy metal, arsenic salt, oxalate, potassium ion, resin, pH, ignition residue measurements, evaluation criterion for allergic reactions, and total solids content of the GLPS injection were all performed using the reference methods in the Chinese Pharmacopoeia. Our results showed that polysaccharide was the key component of injection mixtures. The ignition residue and total solids content in the injection mixture were 1.4% and 2.4%, respectively. The other indices were all within the expected safety ranges. Furthermore, studies from mice functional assays showed that the injection mixture improved the antifatigue capacity of mice without any effect on weight loss/gain. In addition, the injection mixture was safe, which was confirmed by allergy testing in guinea pigs. The development of a GLPS injection offers a novel approach for future medicinal mushroom utilization and holds great commercial promise.