A forward and reverse subtracted cDNA library of cucumber(Cucumis sativus L.) seedling leaves was constructed using suppression subtractive hybridization (SSH) method. The cucumber seedling leaves inoculated with Cladosporium cucumerinum on 2h、8h、20h、32h and72h were used as Tester, and that from untreated leave as Driver. Totally 200 SSH cDNA fragments were selected with Nested primer PCR. Through the DNA sequencing, we got 198 ESTs with high quality. After repeated and redundant sequences removed, 105 ESTs including 50 singletons and 55 contigs were obtained. Protein homology search in non-redundant (Nr) protein database revealed that 17 ESTs didn’t find homologous gene, 88 ESTs were highly homologous with known protein, accounting for 83.8% of all EST sequences, of which only 2 ESTs were unknown function protein. The positive rate of these ESTs was 75.0% detected by DIG Nonradioactive Nucleic Acid Labeling and Detection System. The function of these ESTs involved in energy and basic metabolism, signal transduction, protein and nucleic acid metabolism, photosynthesis and genes specifically induced under certain stress, etc. This study can provide a basis for further study of the cucumber genes resistance to Cladosporium cucumerinum.