Cucumber（Cucumis sativus）inbred line NC-46 contains genes that confer resistance to pathogens including Meloidogyne javanica and Meloidogyne arenaria. Hairy root of NC-46 was induced from cotyledon by wild-type Agrobacterium rhizogenes K599 harboring pBIN-35S-GFP with a frequency of 76.7%. A cDNA library of the hairy root of the transgenic cucumber was constructed successfully. The percentage of recombinant clones in the cDNA library was 98.3%. The capacity of the cDNA library reached 2.02 × 10⁶ cfu，and the percentages of full-length gene and Unigene were 47.6% and 66.7%，respectively. Used of the hairy root of the transgenic cucumber for cDNA library construction overcame the difficulty to obtain a large amount of homogeneous natural roots，the varied physiological states of the roots caused by soil microbes，nematode and RNA contamination. With the modified Smart cDNA construction method，the cloned genes in the cDNA library contain the full length of 5′-introns. Moreover，the cDNAs were inserted into a developed pUC19 plasmid instead of λTripIEx2 phage vector. The cDNA containing plasmid can be used directly for hybridization and sequencing，facilitating gene screening. The cDNA library is useful for the screening of pathogen resistant genes and the cloning of genes involved in hairy root development.