Abstract: In order to establish a specific in situ hybridization method to rapidly batch detect Streptococcus agalactiae from diseased tilapia, a digoxigenin labeled DNA probe was produced by PCR method. The sequence between 132 bp and 599 bp of cfb gene in Streptococcus agalactiae was used as the target sequence. The digoxigenin-labelled cfb probe was verified through sequencing and its length was 430 bp. Meanwhile, the specificity and sensitivity of the probe were also analyzed. Results showed that the probe was able to hybridize with S. agalactiae. And no hybridization signals were observed with other tilapia pathogenic strains (Aeromonas hydrophila, Streptococcus iniae, Plesimonas shigelloides and Staphylococcus aureus), and other homologous Streptococcus strains (Streptococcus bovis and Streptococcus mutans), indicating that the method possessed good specificity. The method could rapidly batch detect Streptococcus agalactiae sensitively, and could detect a template concentration as low as 1.5 ng/μL. Hybridization signals were also observed with other Streptococcus agalactiae from tilapia in Guangdong, Hainan and Guangxi Provinces, and from the diseased tilapia liver and brain. It demonstrated that the DIG-cfb in situ hybridization method possessed the characteristics of rapidity, specificity, sensitivity and could be applied to the batch detection of S. agalactiae from tilapia.