Background <p>Industrial applications of β-glucosidases are often constrained by high salinity, alkaline conditions, and glucose inhibition.</p> Results <p>A glycoside hydrolase family 1 β-glucosidase, B0-BG40, was mined from the metagenome of saline-alkali soil in Karamay, Xinjiang, China. When heterologously expressed in <i>Escherichia coli</i>, B0-BG40 exhibited optimal activity at 45&#xa0;°C and pH 8.6, retaining &gt; 60% of its maximal activity over 20–55&#xa0;°C and pH 5.6–9.6. The enzyme was highly stable at 25&#xa0;°C, 40&#xa0;°C and 45&#xa0;°C and under alkaline conditions, maintaining &gt; 85% residual activity after prolonged incubation and showing activity enhancement following incubation at pH 8.0–10.0. B0-BG40 also tolerated up to 2.0&#xa0;M NaCl and 4.0&#xa0;M glucose, and displayed weak glucose inhibition (<i>K</i>_i = 1033.5 mM). Combined with the results of protein homology modeling and molecular docking, a reasonable mechanistic hypothesis was proposed: the excellent glucose tolerance of the enzyme may be related to its narrow and deeply recessed catalytic channel, and this special channel structure could hinder glucose molecules from entering the active site.</p> Conclusions <p>B0-BG40 is a salt-, alkali-, and glucose-tolerant β-glucosidase with strong potential for applications in food and feed processing and cellulosic ethanol production.</p> Graphical Abstract <p></p>