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One-pot process for the production of ginsenoside Rd by coupling enzyme-assisted extraction with selective enzymolysis was explored in this paper. Several detection methods including HPLC-MS were used to identify and quantify the products in the enzymolysis solution of pectinase. Results showed that ginsenoside Rd was the main component in enzymolysis solution, pectinase specifically hydrolyzes protopanaxadiol (PPD)-type ginsenoside and was a selective enzyme to convert ginsenoside Rb1 to Rd in a way. In addition the influencing factors on the yield of ginsenoside Rb1 and Rd were optimized using L 9 (3 4) orthogonal design data. The enzymolysis conditions for the higher yield of Rd were 52.5 °C, pH 6 and 1 h with a yield of 0.8314 from 50 mg drug material. The controllable transformation hypothesis of the PPD-type ginsenoside was also explored from the perspective of the molecular steric hindrance. Pectinase could be used as an efficient enzyme for one-pot producing ginsenoside Rd.