The population growth and the search for new industrial applications of the generated by-products in food industries have motivated several research efforts, aiming its destination for microorganism’s development to obtain microbial biomass, in which some of these microbial sources may provide health benefits to the consumer. In this context, this work evaluated the influence of pH variation on the microbial biomass cultivation in the fermentation process of the whey permeate by using the Saccharomyces boulardii probiotic yeast. The results showed that the pH value significantly influences the biomass production, wherein the highest production of probiotic biomass (10.45 g L-1) was achieved at the pH 4.5. In general, the cheese whey permeate may be a feasible alternative source for probiotic biomass cultivation.