A study with two cross-sectional surveys in two consecutive years was conducted in Shanghai, China to examine the seroprevalence of herpes simplex virus type 2 (HSV-2), syphilis and HIV among female sex workers (FSW). A total of 793 FSW participated in the survey, 382 in 2008 only, 382 in 2009 only, and 29 in both 2008 and 2009. The majority of them were less than 30 years and two-thirds were married. All were migrants and a half was from rural areas. Some of them have stayed in Shanghai and engaged in commercial sex for more than two years. Their knowledge of HIV/AIDS was limited. Condom use was not common for both marital sex and non-commercial extramarital sex but fairly frequent for commercial sex. Two-percent were using drugs in the past year. Nearly one quarter reported having syndromes of sexually transmitted diseases (STD) in the past year, with a substantial proportion of them untreated or treated inappropriately. No one was HIV-infected. The overall HSV-2 seroprevalence was 47.3% (375/793), 45.5% (187/411) in 2008 and 50.1% (206/411) in 2009. The overall prevalence of syphilis was 4.5% (36/793), 7.0% (29/411) in 2008 and 2.4% (10/411) in 2009. Multiple logistic regression analyses indicated that HSV-2 infection was statistically associated with age, type of working venue, origin, and period of staying in Shanghai; whereas syphilis infection was statistically associated with year of participation and smoking status. In conclusion, HSV-2 is highly prevalent among FSW in Shanghai, with a relatively low prevalence of syphilis. The currently low HIV epidemic in this population provides a window of opportunity to implement tailored HIV/STD prevention and control efforts targeting them, with particular attentions to large numbers of HSV-2-infected FSW and their clients.