Abstract: We extracted the total RNA of Vibrio harveyi-challenged abalone (Haliotis diversicolor Reeve).First and second strand cDNA were synthetized by using Creator SMART cDNA Construction Kit and further homogenization of cDNA library was carried out with duplex-specific nuclease (DSN) treatment. Fragments at length of 0.5~1.0 kb and 1.0~3.0 kb were ligated to pDNR-LIB and transformed into Esherichia coli strain DH10B. Both two cDNA libraries have a capacity of 2.5×10⁵ cfu · mL^-1, and all of the 30 randomly picked colonies are identified as positive recombinant plasmids by PCR. Furthermore, 200 randomly selected clones were sequenced and 174 high quality ESTs were acquired, which belong to 149 unigenes including 12 contigs and 137 singlets after assembling with a redundancy rate of 14.37%. The result indicates that 39 unigenes get high similarity with known genes. In conclusion, the cDNA library we have constructed has good quality and can facilitate further research work.