The clinical and laboratory analysis of 380 urogenital tract secretion samples from sexually transmitted diseases patients for Chlamydia trachomatis detection was performed by polymorphonuclear leukocyte count and fluorescent real-time polymerase chain reaction (PCR). The results showed that of 216 male patients 92.6% presented urethral polymorphonuclear leukocyte count ≥5, and 62 cases (28.70%) were Chlamydia trachomatis-positive detected by PCR. There were 30 case with Chlamydia trachomatis and Neisseria gonorrheae coinfection (13.98%). Of 164 female patients, 53.0% presented cervical canal polymorphonuclear leukocyte count ≥10, and 33 cases (20.1%) were Chlamydia trachomatis-positive detected by PCR. The results indicate that the application of real-time fluorescent PCR can improve positive detection rate of Chlamydia trachomatis and control its spread in sexually transmitted disease clinics.