A high quality cDNA library from the venom gland of Chinese Gloydius shedaoensis shedaoensis was constructed using SMART(switching mechanism at 5′ end of RNA transcript) technique.Total RNA was extracted using RNAiso reagent kit and mRNA was purified using Poly(A) Purist^TM kit,respectively.Singlestrand cDNA was synthesized using reverse transcription PCR and double-strand cDNA was synthesized and amplified using LD(long-distance) PCR.The cDNA fragments with the size over 400 bp were fractionated and ligated to pDNR-LIB vector.Recombined plasmids were then elelctrotransformed in Escherichia coli DH10B strains.The capacity of the constructed library was 8 × 10⁷ cfu /ml.The size of most inserts was larger than 1 000 bp.The percentage of the selected recombinant clones was very high(100%).Thus,a cDNA library from the venom gland of G.s.shedaoensis has been successfully constructed.It has a high recombinant expression level,and can be used for cloning and expressing bioactive protein genes from G.s.shedaoensis and for studying their biological functions.