2-Selenouridine (SeU) is one of the naturally occurring modifications of Se-tRNAs (SeU-RNA) at the wobble position of the anticodon loop. Its role in the RNA-RNA interaction, especially during the mRNA decoding, is elusive. To assist the research exploration, herein we report the enzymatic synthesis of the SeU-RNA via 2-selenouridine triphosphate (SeUTP) synthesis and RNA transcription. Moreover, we have demonstrated that the synthesized SeUTP is stable and recognizable by T7 RNA polymerase. Under the optimized conditions, the transcription yield of SeU-RNA can reach up to 85% of the corresponding native RNA. Furthermore, the transcribed SeU-hammerhead ribozyme has the similar activity as the corresponding native, which suggests usefulness of SeU-RNAs in function and structure studies of noncoding RNAs, including the Se-tRNAs.