Photoaffinity labeling is widely applied to demonstrate targets of small molecule ligands. In this paper, biotin photoaffinity labeled molecule with propargyl group 1 has been designed and synthesized, followed it's labeling of N2-acetyl-2'-O-propargyl guanosine 9 by "click chemistry". This technology presents delight development potential in labeling of second messenger cyclic nucleotide, antisense oligonucleotide or siRNA.