In this work, we addressed these obstacles by adjusting the reaction conditions to accommodate for the solubility and stability of Se-nucleobases in the biocatalytic synthesis of 2-Se-pyrimidine nucleosides. Working under hypoxic conditions prevented deselenation and oxidative loss of yield while an increased reaction pH allowed for higher substrate loading enabled by deprotonation of the Senucleobases. A thermodynamic characterization of the reaction equilibria via analytical transglycosylations revealed equilibrium constants well outside the known range of similar nucleosides and guided our choice of suitable reaction conditions to maximize the product yields. This report presents improved yields compared to established procedures and describes the first biocatalytic synthesis of 2-Se-pyrimidine nucleosides.