Streptococcus agalactiae (GBS) is the major contagious cause of early-onset sepsis in newborns. Screening and intrapartum antibiotic prophylaxis for GBS in pregnant women could effectively prevent early-onset GBS infection in newborns. The conventional method for isolation and identification of GBS on the blood plate medium is labor-intensive, time-consuming, and low sensitive. This study is aimed to optimize parameters for a realtime PCR reaction with primers and a probe designed to detect GBS-specific cfb gene. The optimized experiments were carried out on the strain S. agalactiae ATCC 13813. The specificity of the optimized procedure was tested on DNA samples of Staphylococcus aureus ATCC 25923, Gardnerella vaginalis, and Chlamydia trachomatis. In addition, the optimized reaction was tested on 30 vaginal - rectal samples from pregnant women between 35-37 weeks gestation. The optimized procedure was specific to GBS with 50 copies/reaction sensitivity, an accuracy of 99.94%, and amplification efficiency (EA%) of 94.5%. GBS was detected in ten samples among the 30 vaginal-rectal samples by the realtime PCR, while only eight samples were found to be positive in the conventional plate method.