In order to obtain a quality subtractive cDNA library of age-related cataract which contains more large fragments from small amounts of sample, an improved subtractive hybridization which using magnetic beads isolation and biotin labelling was used to obtain the subtractive cDNA. Then the large fragments of the subtractive cDNA were amplified by using selective PCR method, so the subtrative cDNA library of age-related cataract was successfully constructed. 22 clones of the library were selected at random and were identified by the restriction enzymes digestion. 7 fragments were larger than 1 000 bp,which is 31.8% of the totals, 15 fragments were larger than 750 bp, which is 68.2% of the totals. The cDNA fragments are larger and can be used in further study. A subtractive cDNA library with high quality and more large fragments can be constructed rapidly and effectively by improved subtractive hybridization and selective PCR method from small amounts of sample.