This study aimed to obtain yeast strains with strong selenium-enriching capacity and to isolate bioorganic selenium from them for preparing selenium-enriched foods. Microbial isolation techniques, selenium tolerance screening and the tellurite reduction assay were employed. A yeast strain with excellent selenium metabolism characteristics was selected from 12 strains isolated from selenium-enriched soil or fruit peel samples and was identified as Pichia kudriavzevii. After ultraviolet mutagenesis and fermentation condition optimization, the selenium accumulation in this strain increased to 1678 mg/L, with a selenium conversion rate of 98.6%. Finally, the yeast cells were lysed by high-pressure freeze disruption before the extraction of bioorganic selenium, which was incorporated into yogurt and biscuits to produce selenium-fortified foods. This study provides technical references and a data foundation for developing selenium-enriched microbial resources and their application in functional products.