The cyclodextrin glycosyltransferase (CGTase) was used to catalyze the conversion of starch into cyclodextrins (CD) in industry. Improving the activity of CGTase to produce more CD with relative low cost is intensely interesting and has drawn wide attention. Amino acid mutation of His167 into Cys significantly enhanced β-CGTase activity; however, optimization of culture conditions for β-CGTase-H167C remains unclear. To determine this, the medium and culture conditions for β-CGTase-H167C were optimized with response surface methodology. Maximum activity of β-CGTase-H167C was obtained with the medium containing 1.1% corn starch, 4.4% corn steep liquor, 1.1% peptone, 0.02% MgSO·7HO and 0.1% KHPO·3HO that were cultured with the initial pH 8.4, incubation temperature at 37.4 °C, with 5% inoculation size and shaking speed at 202 r/min. Under the optimal conditions, the activity of β-CGTase-H167C was up to 4355 U/mL, which is 1.93-fold in comparison with the initial activity. Our results established the promising culture strategy for the production of cyclodextrins by β-CGTase-H167C.