Objective To explore syphilis infection and its influencing factors among older female sex workers (OFSWs) in Guigang City, provide scientific theoretical basis for making effective measures and strategies for prevention and control of syphilis among this population. Methods In 2010-2016, a sentinel sampling survey among female sex workers (FSWs) in Guigang City was performed according to the "National AIDS Sentinel Surveillance Program", a face-to-face anonymous survey on subjects was conducted by trained investigators using "Questionnaire of National AIDS Sentinel Surveillance on FSWs", and blood specimens were taken to detect syphilis and other infections. Results A total of 2 851 OFSWs in Guigang City were investigated, the main sources of OFSWs were marriage/cohabitation, household registration in other provinces, Han nationality, primary education, and low-grade locations, the median age was 41 years old (interquartile range:38-45). Among 2 851 OFSWs, the positive rates of syphilis, HIV, and HCV were 17.33%, 2.84%, and 1.89% respectively; 5.02% of OFSWs were diagnosed with sexually transmitted diseases (STDs) during the last year, 88.46% used condoms in the latest commercial sexual intercourse, and 57.42% used condoms every time during the latest month commercial sexual intercourse. 87.93% and 34.86% of OFSWs respectively received condom publicity and distribution/AIDS counseling and detection as well as peer education intervention services. Multivariate analysis showed that low grade locations, Zhuang nationality, illiteracy, age ≥ 35 years, without receiving condom publicity and distribution/AIDS counseling and detection intervention services, as well as positive for HIV and HCV antibody detection were independent risk factors for syphilis infection in OFSWs. Conclusion Infection status of syphilis among OFSWs in Guigang City is severe, low grade locations, Zhuang nationality, illiteracy, age ≥ 35 years, without receiving condom publicity and distribution/AIDS counseling and detection intervention services, positive for HIV and HCV antibody detection are high risks for syphilis infection in OFSWs. It is necessary to strengthen intervention services such as condom publicity and distribution, increase condom use among OFSWs, and adopt comprehensive prevention and control measures against sexually transmitted diseases such as syphilis, HIV and HCV.