The present paper aims to investigate the antimicrobial activity of tetracycline against clinical Streptococcus agalactiae (S. agalactiae) isolates. A total of 136 S. agalactiae isolates were collected retrospectively in Nanshan People’s Hospital between 2014 and 2017. The minimum inhibitory concentrations (MICs) of tetracycline were determined by agar dilution. Polymerase chain reaction (PCR) was used to detect Tet-specific resistance factors and housekeeping genes. The strains were typed by multilocus sequence typing (MLST). The results showed that the resistance rate of S. agalactiae isolates to tetracycline was 46.32% (63/136), and the tetracycline-resistant strains mainly carried tetM. The dominant ST types were ST17 and ST19 strains. The antimicrobial activity of tetracycline against ST17 strains was significantly higher than ST19 strains. It is concluded that the tetracycline resistance in S. agalactiae isolates is associated with tetM gene and the dominant strains are ST17 strains and ST19 strains.