The present study aimed to investigate the effect of oleaster flour on Lactobacillus acidophilus, L. delbrueckii subsp. bulgaricus, and Bifidobacterium animalis subsp. lactis in probiotic yogurt during its storage period and whether oleaster flour has a protective effect against gastric fluid for these probiotic bacteria. For that purpose, the effect of oleaster flour at different doses (1%, 2%, and 3% w/v) on the titratable acidity, pH, and microbiological properties was investigated throughout cold storage. In addition, on the first day of storage, in vitro tolerance of probiotics in pH adjusted to (pH 2.0-ph 4.0) simulated gastric fluid was investigated for 1, 60, 120, and 180 min. Yogurt with a higher dosage (2%-3%) of oleaster flour had a higher pH and lower titratable acidity. Moreover, the addition of 3% oleaster flour showed a preservative effect on L. acidophilus, L. delbrueckii subsp. bulgaricus and B. animalis subsp. lactis during storage. On the first day of storage in pH 4.0 for synthetic gastric fluid, in vitro acid tolerance of all probiotics showed stability for 180 minutes. Also, at pH 2.0 SGF, B. animalis subsp. lactis was below the detectable limit in the control and 1% of groups. However, the 2% and 3% groups showed nearly 3 log cfu/g viability at the end of 180 min. These positive effects were related to the buffering effect of the oleaster peel. Thus, these results could prove that oleaster flour can be used for the production of bio-yogurt.