Biosurfactants are a family of diverse amphipathic molecules that are produced by several microorganisms such as bacteria, molds, and yeasts. These surface active agents have several applications in agriculture, oil processing, food, and pharmaceutical industries. In this research using YMG and YUG culture media, a native yeast strain, HG5, was isolated from honey bee. The oil spread test as a screening method was used to evaluate biosurfactant production by the yeast HG5 isolate. The 5.8s-rDNA analysis confirmed that the isolated yeast was related to Lachancea thermotolerans. We named this strain Lachancea thermotolerans strain BBMCZ7FA20 and its 5.8s-rDNA sequence was deposited in GenBank, NCBI under accession number of KM042082.1. The best precursor of biosurfactant production was canola oil and the sophorolipid amount was measured for 24.2 g/l. The thin layer chromatography and Fourier Transform Infrared Spectroscopy analysis showed that the extracted biosurfactant from Lachancea thermotolerans was sophorolipid. In conclusion, this is the first report of sophorolipid production by a native yeast Lachancea thermotolerans BBMCZ7FA20 we isolated from the honey bee gut collected from an apiary farm in Saman, Chaharmahal Bakhtiari province, Iran. We suggested that some cost-effective supplements such as canola oil, sunflower oil, and corn oils could be applied for increasing the sophorolipid production by this native yeast strain. According to several applications of biosurfactants in today world, the production of sophorolipid by Lachancea thermotolerans could be considered as a potential in the current industrial microbiology and modern microbial biotechnology.