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Our previous studies have revealed that the Tricholoma lobayense polysaccharide TLH-3, which has an IC50 value for scavenging superoxide radicals comparable to that of Vitamin C (VC), could be prepared by hot water extraction and column chromatography. However, this method is tedious and inefficient. In this study, high-pressure homogenization (HPH) was used to extract polysaccharides from T. lobayense Heim. The yield of 17.3% using HPH was higher than the 12.3% yielded by traditional extraction. Moreover, the operation combined quaternary ammonium salt precipitation with ultrafiltration (QASP-UF) to purify the crude polysaccharides successfully, which provided a higher TLH-3′ yield, a larger separation volume, and a shorter purification time than that of column chromatography. The whole process for TLH-3′ preparation presented here exhibited significant advantages of quicker processing, low consumption, high efficiency, and flexible compatibility with follow-up studies. Based on a characterization of molecular weight, Fourier Transform Infrared Spectroscopy (FT-IR) analysis, and monosaccharide composition analysis, it can be concluded that TLH-3 and TLH-3′ are the same polysaccharide. Antioxidant activity was tested to prove that the TLH-3′ prepared in this study could scavenge DPPH and superoxide radicals, with IC50 values of 111.7 μg/mL and 160.0 μg/mL, respectively. Its outstanding antioxidant ability was comparable to that of ascorbic acid. [ABSTRACT FROM AUTHOR]