Background Heterosexual contact is the primary mode of HIV transmission in China and commercial sex is thought to play a crucial role in China’s epidemic. Female sex workers (FSWs) in China tend to be either brothel-based (BSWs) or street-based (SSWs), but few studies have investigated the differences between these important segments of this difficult-to-reach, high-risk population. Our aim was to explore the differences between SSWs and BSWs in terms of socio-demographic characteristics, sexual and risky practices, HIV/STI-related knowledge, health services, HIV/STI prevalence and other aspects. Methods A cross-sectional survey was conducted in Yunnan Province of China in partnership with a local FSW-friendly non-governmental organization. Face-to-face interviews using a structured questionnaire were conducted to collect data on socio-demographic characteristics, sex work history, sexual behaviours, HIV/STI-related knowledge, HIV testing history, and healthcare services uptake. Blood samples were taken for HIV and syphilis testing, and urine samples for gonorrhea and chlamydia testing. Descriptive statistics were used to evaluate differences between SSWs and BSWs. Results A total of 185 BSWs and 129 SSWs were included in the study. SSWs were older and less educated, had more dependents and more clients, lower condom use and accessed fewer healthcare services. Moreover, 37.2% of SSWs and 24.9% of BSWs were found to have HIV/STI infection. Unfortunately, the awareness related to STIs was relatively low in both groups, especially SSWs. Conclusions Our study provides evidence that confirms the disproportionately high vulnerability of SSWs to HIV and other STIs, underscoring the urgent need for the Chinese health and public health sectors to prioritize outreach to SSWs. Awareness and educational programs, condom distribution, testing and health check-ups should be included in a comprehensive strategy for HIV/STI prevention in this high-risk population.