Abstract: In order to construct a full-length cDNA library of bitter melon fruit in goods mature period and analyze the sequence of EST, in this research, we constructed a full-length cDNA library of bitter melon fruit for polypeptide bitter melon parent‘189-4’in goods mature period using the SMART technology. The tested results showed that the capacity of the library was 2.08×106, restructuring at a rate of 96.29%, the average insertion fragment ranged from 750 to 2000 bp, and the insert fragment average size was about 1000 bp. By sequenced 400 monoclonal randomly, we got 370 EST and the sequence analysis results showed that 350 had a homology, full length rate was 70%; 19 sequence had no matching, 320 was unigene, among them there were 10 contigs, 260 single genes, and there were about 48 genes possibly related to the nutrition quality. This research laid a foundation for the screening, cloning of functional genes for the bitter melon.