Abstract: To systematically evaluate the effects of steam explosion(SE) pretreatment on the chemical composition and the anti-glycation and antioxidant activities of Tuber melanosporum extract, extracts obtained by hot reflux extraction from SE-treated samples were investigated. Changes in chemical composition and in vitro functional properties were analyzed. A bovine serum albumin–glucose (BSA–Glu) non-enzymatic glycation model, together with multiple free radical scavenging assays, was established to compare the anti-glycation and antioxidant activities of extracts before and after SE treatment. The results showed that SE pretreatment significantly increased the total polysaccharide content by 20.44%, while the protein content decreased by 38.14% (P<0.01). In the BSA–Glu system, the extract effectively inhibited the formation of fructosamine, reactive dicarbonyl compounds (methylglyoxal and glyoxal), and fluorescent advanced glycation end products (AGEs), and significantly reduced the accumulation of protein oxidation products, including dityrosine, kynuurine, and N'-formylkynuurine(P<0.05). Compared with the untreated sample, the anti-glycation activity of the SE-treated extract was significantly enhanced. In addition, SE pretreatment markedly improved the antioxidant activity of the extract (P<0.01), with IC₅₀ values of 0.96±0.04、0.50±0.02 and 2.98±0.03 mg/mL for DPPH·、·OH, and Fe²⁺ scavenging assays, respectively. In conclusion, Tuber melanosporum extracts exhibits notable anti-glycation and antioxidant activities, and SE pretreatment effectively enhances its in vitro bioactivity, providing a scientific basis for the functional development and utilization of Tuber melanosporum resources.