Selenium-modified nucleosides are powerful tools to study the structure and function of nucleic acids and their protein interactions. The wide-spread application of 2-seleno pyrimidine nucleosides is currently limited by low yields in established synthetic routes. Here, we describe the optimization of the synthesis of 2-Se-uridine and 2-Se-thymidine derivatives by thermostable nucleoside phosphorylases in transglycosylation reactions using natural uridine or thymidine as sugar donors.