In this study, the effect of stevia and inulin interactions on fermentation profile of Lactobacillus acidophilus in milk and in vitro systems was investigated. The medium were analysed for pH, cell density (OD600), probiotic bacterial counts, prebiotic activity score (PAS), lactic acid and short-chain fatty acid (SCFA) content. In the second stage of the study, the potential prebiotic activity of stevia and its effect on milk fermentation characteristics as sugar substitute were evaluated in symbiotic milk system. Consequently, it was found that stevia improved the survival of L. acidophilus in in vitro and in the milk matrix and could be assigned as a potential prebiotic source and sugar replacer for the manufacture of sugar-reduced dairy products. These results show that the in vitro-positive prebiotic activity of stevia is closer to inulin and is related to its fermentation profile and growth parameters with high short-chain fatty acid production. The counts of probiotic bacteria remained within the biotherapeutic level (>7 log(10) cfu/mL) during 28 days of storage in stevia and inulin milk system with high viscosity index and gel characteristics.