It relates to a method for preparation of four kinds of 2′, 3′-cNMPs (2′, 3′-cAMP, 2′, 3′-cGMP, 2′, 3′-cCMP and 2′, 3′-cUMP), comprising steps of: (1) extract genomic DNA and amplify gene If3; (2) ligate If3 gene to expression plasmid to construct a recombinant vector, and transfer the recombinant vector to E. coli to obtain a recombinant strain. Cultivate the recombinant strain and collect the fermentation broth; (3) collect the cells form the fermentation broth and disrupt the cells, and then purify the recombinant protein IF3 from the cell extract by Ni2+-nitrilotriacetic acid resin. Incubate the recombinant protein IF3 solution at 0° C. for 3 days to release 2′, 3′-cNMPs from IF3, and centrifuge the solution; (4) Ultrafiltrate the supernatant to remove proteins, and prepare four kinds of 2′, 3′-cNMPs by high-performance liquid chromatographic (HPLC) on a C18 reversed-phase column.