The objectives of this study are isolate of biotingenic yeast from different sources, showing the ability of isolated yeast in biotin production and carry out molecular characterization and differentiation between biotingenic and non¬¬-biotingenic strains. Infested specimens of samples of Malus domestica, Vitis vinifera, Protulaca oleracea, Mangifer indica, and Eruca sativa, were collected from local market in sterile plastic bags and then used to isolate the test microorganisms. Yeast extract agar plates were inoculated by moistened swab then incubated at 30°C for 48 hours. Three types of microorganisms were isolated and identified as Sporobolomyces roseus, Sporobolomyces linderae and Candida pararugsoa. The isolated microorganisms were subjected to produce biotin using Feed-Batch technique by flasks containing 50ml yeast extract peptone dextrose broth medium (YEPD). All flasks were incubated in shaker incubator at 30°C for one week. Biotin determined and measured by flourometrics techniques using 400-500 nm wavelengths. The concentrations were ranged from (148.03 mg/l and 0.00095 mg/l) which obtained by S.roseus and S.linderae respectively. Candida pararugosa showed as non-biotinogenic microorganism. This study was concluded that the S.roseushas ability to produce biotin in significant amount (1.48g/l) with cheep substrate and low cost, in one week production, and at pH 6.0.