Hydroxynitrile lyases (HNLs) are increasingly finding application in the synthesis of enantiomerically pure cyanohydrins which are important intermediates in the production of pharmaceuticals and agrochemicals. Synthesis of enantiopure mandelonitrile was carried out using HNL of wild apricot (Prunus armeniaca L.) [ParsHNL] in aqueous/organic biphasic system. The optimum pH and temperature of the reaction were 4.0 and 15 degrees C respectively, which are important parameters to suppress the non-enzymatic catalysis. ParsHNL catalyses synthesis of (R)-mandelonitrile in methyl-tbutyl ether (MTBE)/citrate buffer biphasic system with > 99% ee. Synthesis of mandelonitrile was carried out in batch reaction at 40 ml scale and finally 2.7 mmoles of (R)-mandelonitrile was recovered which corresponded to 90% molar conversion in 46 h reaction. In fed batch reaction 6.37 mmoles of (R)-mandelonitrile could be produced which corresponds to 91% molar conversion in 46 h. In both reactions, enzyme produces (R)-mandelonitrile with > 99% ee which showed enhanced selectivity as compared to aqueous reaction (96% ee) by ParsHNL. The results showed potential of ParsHNL to synthesize (R)-mandelonitrile in both, batch reaction and fed-batch reaction and can be effectively used in the synthesis of (R)-mandelonitrile.