The synthesis of optically active (R)-ketone-cyanohydrin catalyzed by (R)-oxynitrilase from Prunus salicina seed meal via enantioselective transcyanation of acetyltrimethylsilane with acetone cyanohydrin in the water/organic solvent biphasic system was studied by GC analysis on a chiral β-Cyclodextrin Column. The effects of different (R)-oxynitrilase sources, diameter size of crude enzyme, substrate concentration, the ratio of two substrates, enzyme concentration and substrate structure on the enzymatic enantioselective transcyanation were investigated systematically. The results showed that (R)-oxynitrilase from Prunus salicina could efficiently catalyze enantioselective transcyanation of acetyltrimethylsilane with acetone cyanohydrin. The optimal diameter size of crude enzyme, concentration of acetyltrimethylsilane, ratio of acetone cyanohydrin to acetyltrimethylsilane and crude enzyme concentration were 0.3～0.45 mm, 21 mmol/L, 2∶1 and 60.9 g/L, respectively. (R)-oxynitrilase from Prunus salicina could not accept 3, 3-dimethyl-2-butanone as substrate, while both high substrate conversion and high product ee% were obtained with its silicon counterpart acetyltrimethylsilane as the substrate. Under the optimal conditions, both acetyltrimethylsilane conversion and enantiomeric excess of the product were above 99%. These results demonstrated that the silicon atom in substrate served as a more effective atom than the carbon atom to enhance the activity of this enzyme.