Abstract: To screen the specific genes of Streptococcus agalactiae induced in Oreochromis niloticus, the genomic expression library of the virulent strain Hod-1 was constructed. The Sau3AⅠ was used to digest genomic DNA of S.agalactiae strain Hod-1 incompletely. The DNA fragments ranging from 0.5 kb to 2 kb were recycled and ligated into pET-28 a/b/c expression vectors which had been digested by BamHⅠ and dephosphorylated by SAP before using. The recombinant vectors were transformed into BL21 (DE3). The results show that the genomic expression library of S.agalactiae contained 1.024×10⁵ clones, considerably more than the minimum number of clones (45 579 clones) covering the whole genomic DNA of S.agalactiae. Results of PCR detection show that the rate of fragment insertion was 94% and different sizes of inserted fragments ranged from 0.5 kb to 2.0 kb. The results of sequencing show that the homology between inserted sequences and the genomic DNA sequnce of S.agalactiae was over 99%. In conclusion, the genomic expression library of S.agalactiae strain Hod-1 was constructed successfully, which can be used for screening genes of S.agalactiae induced expression in O.niloticus.