The potential use of ram horn hydrolysate (RHH) as a supplement for enhancement of glycerol production by Saccharomyces cerevisiae was studied. For this purpose, first, RHH was produced. Ram horns were hydrolyzed by treating with acid (6 N H2SO4), and the RHH was obtained. The contents of protein, nitrogen, ash, some minerals, total sugars, total lipids and amino acids of RHH were determined. With the addition of RHH to the fermentation medium with a final concentration of 4% (optimal concentration), the glycerol value reached a maximum value of 9.4 gl(-1), which is 47% higher than that of the control experiment. The addition of 4% (v/v) RHH enhanced the glycerol accumulation, reduced the residual sugar concentration and stimulated yeast growth. Adding 4% RHH had no adverse effects on the cells of S. cerevisiae. As a result, RHH was found to be suitable as a valuable supplement for glycerol production in the batch fermentation.