Rebaudioside D is a promising sweetener due to its zero calorie and high sweetness. Here, a transglucosylase gene eugt11 from Oryza sativa was for the first time expressed in Pichia pastoris, and transformant XE-3 showed the highest expression levels in pH 5.5 BMMY media containing 0.75% methanol. The affinity-purified EUGT11 from XE-3 displayed the highest activity at pH 6.0-6.5 and 45 °C, compared to pH 8.5 and 35 °C for EUGT11 from Escherichia coli. One-pot synthesis with orthogonal design was employed to optimize the rebaudioside D production using XE-3, and the initial pH 7.0 of the medium appears to be a significant factor and delivers the highest conversion efficiency. A two-step temperature-control strategy was developed, and a conversion rate of 95.31% was achieved at 28/35 °C vs. 62.41% in a one-step process at 28 °C. This study provides a high-efficient whole-cell biocatalysts technology for the sweetener production.