Biotin-labeled RNA has found broad applications in chemistry, biology and biomedicine. In this protocol, we describe a simple procedure for 5′ RNA biotin labeling by one-step in vitro transcription. A biotin–AMP (adenosine 5′-monophosphate) conjugate, biotin-HDAAMP (adenosine 5′-(6-aminohexyl) phosphoramide; where HDA is 1,6-hexanediamine), is chemically synthesized. Transcription initiation by biotin-HDAAMP under the T7 φ2.5 promoter produces 5′ biotin-labeled RNA with high labeling efficiency. The procedure is especially useful for biotin labeling of RNA that is larger than 60 nucleotides. In addition, the protocol provides an attractive alternative to chemical synthesis of biotin-labeled small RNA of less than 60 nucleotides, particularly when the desired quantity of RNA is low. The whole procedure, from chemical syntheses to isolated biotin-labeled RNA, can be completed within 2 weeks.