The deep eutectic solvents (DESs) + K₂HPO₄ aqueous two-phase systems were prepared. The phase separation capacity of betaine–glycerol became weakened with the increase in temperature, followed by choline chloride–glycerol and betaine–glycol (Bet–EG). However, the temperature did not have a significant effect on the biphasic region for ChCl–EG. β-glucosidase with the tag of choline-binding protein (LytA) was constructed and expressed successfully, and purified by APTSs with enhanced enrichment based on the affinity adsorption between choline salt and choline-binding protein. In the ATPS composed of 23.11% Bet–EG and 40.50% K₂HPO₄, the recombinant β-glucosidase (GLytA) has the best purification effect with a recovery rate of 89.07% and purification factor of 8.3359. Furthermore, when the purified GLytA was mixed with cellulase to catalyze microcrystalline cellulose, the saccharification rate was up to 81.2%. The activity of the mixed enzyme did not decrease significantly in the 8 days. There was still 64.22% residual activity in the mixture after 35 days. The purified recombinant enzyme by a single step can be used for subsequent catalysis or preservation directly, which has the potential to applicate in industry.