Rhamosidase producing yeast strain 84 was isolated from whey beverage and identified as Clavispara lusitaniae KF633446. The effect of different carbon sources (rhamnose, glycerol, lactose, fructose, glucose and sucrose), nitrogen sources (yeast extract, peptone, ammonium chloride, ammonium sulphate, urea and casein), temperature (10-60°C) and pH (3-8) were studied to optimize the production of rhamnosidase enzyme from Clavispara lusitaniae 84. Further, a multivariate response surface methodology evaluated the effects of different factors on enzyme activity and optimized enzyme production. The fit of the model (R2 = ) was found to be significant. | Optimization of media components for production of α-L-rhamnosidase from clavispora lusitaniae KF633446