Abstract: This study aimed to obtain a highly efficient expression of cyclodextrin glycosyltransferase (CGTase) in Pichia pastoris. The optimized CGT2 was cloned into a yeast constitutive expression vector, pGATZαA.The recombinant plasmid pGAPZαA-CGT2 was then transformed into P. pastoris by electroporation to construct an engineered strain, X33/pGAPZαA-CGT2.After cultivating for 120 h in a shaking flask, CGT2 with an activity of 0.21 U·mL^-1 was obtained. Experiments were conducted to further optimize the fermentation conditions. As a result, the greatest activity of 1.26 U·mL^-1, achieving a 1.7-fold improvement, for the enzyme was reached by induction for 120 h at pH 6.5 and 28℃ with a constant shaking at 200 r·min^-1 and replenishing with 2.5% glycerol every 24 h in the flask.