The telomerase activator cycloastragenol (CA) is regarded as a potential anti-aging drug with promising applications in the food and medical industry. However, one remaining challenge is the low efficiency of CA production. Herein, we developed an enzyme-based approach by applying two enzymes (beta-xylosidase: Xyl-T; beta-glucosidase: Bgcm) for efficient CA production. Both key glycosidases, mined by activity tracking or homology sequence screening, were successfully over-expressed and showed prominent enzymatic activity profiles, including widely pH stability (Xyl-T: pH 3.0-8.0; Bgcm: pH 4.0-10.0), high catalytic efficiency (k(cat)/K-m: 0.096 mM(-1)s(-1) (Xyl-T) and 3.08 mM(-1)s(-1) (Bgcm)), and mesophilic optimum catalytic temperature (50 degrees C). Besides, the putative catalytic residues (Xyl-T: Asp311/Glu 521; Bgcm: Asp311/Glu 521) and the potential substrate-binding mechanism of Xyl-T and Bgcm were predicted by comprehensive computational analysis, providing valuable insight into the hydrolysis of substrates at the molecular level. Notably, a rationally designed two-step reaction process was introduced to improve the CA yield and increased up to 96.5% in the gram-scale production, providing a potential alternative for the industrial CA bio-production. In essence, the explored enzymes, the developed enzyme-based approach, and the obtained knowledge from catalytic mechanisms empower researchers to further engineer the CA production and might be applied for other chemicals synthesis.