Lactobacillus acidophilus isolated indigenously from Eleusine coracana has been reported by us to be a good producer of beta-galactosidase (Akolkar et al., 2005). Recovery of this intracellular enzyme using ultrasonication was found to be the rate-limiting step for subsequent downstream processing. Among the various chemical and mechanical methods tried, ultrasonication was found to be the most effective. However, prolonged ultrasonication can result in partial to complete degradation of the released enzyme. This article reports the optimization of the ultrasonication process for release of beta-galactosidase using response surface methodology (RSM). Factors like cell density, sample volume, and ultrasonication time, which were critical for the release of the enzyme at a constant acoustic power, were considered in the optimization to develop an efficient release process. Increase in the release of beta-galactosidase from 320U/gm DCW to 483 +/- 1.9U/gm DCW was achieved at the optimized ultrasonic process conditions (cell density 2.1% wet weight, suspension volume 11 mL, and ultrasonication time 15 min) at a constant acoustic power of 50 W.