Background: Syphilis remains a significant global public health issue, and female sex workers (FSWs) are highly vulnerable to the etiological agent of this disease. This study aimed to describe the prevalence of exposure to Treponema pallidum, as well as the vulnerability factors among FSWs in the state of Pará, Brazilian Amazon. Methods: A cross-sectional, retrospective study involving 360 FSWs from five cities in Pará was conducted from 2005 to 2007. Blood samples were collected for treponemal and non-treponemal testing, and epidemiological information was obtained through interviews. Results: The exposure rate to T. pallidum was 37.7% (136/360), and the majority of FSWs had serological results indicating past exposure (21.1%). Among the FSWs exposed to T. pallidum, most of them were single, aged 23 to 42 years old, had less than 8 years of schooling, and had a family income of between 1 and 3 minimum wages. They reported using condoms during sexual intercourse and had no history of sexually transmitted infection (STI). Furthermore, many of the FSWs exposed to T. pallidum reported having more than 20 sexual partners per month, and had partners from other Brazilian states, but not from other countries. An age over 42 years and a reduced level of education were factors associated with exposure to T. pallidum. Finally, a high rate of exposure to T. pallidum among FSWs in the Brazilian state of Pará (from 2005 to 2007) was detected. In later years, epidemiological studies conducted with FSWs recorded that this rate remained high. Measures to control, treat, and prevent syphilis among FSWs were necessary between 2005 and 2007, and they are still imperative today. Actions related to educational programs and STI control, treatment, and prevention measures contained in Brazilian policies aimed at women’s health have not changed the vulnerability scenario of FSWs regarding their exposure to T. pallidum, even after 16 years, and must be reviewed and adapted to the conditions of the Brazilian Amazon.