Objective To understand the awareness rate of acquired immune deficiency syndrome (AIDS) prevention knowledge, behavioral characteristics, and the prevalence of human immunodeficiency virus (HIV), syphilis and hepatitis C virus (HCV) among female sex workers (FSW) in Anhui Province from 2010 to 2024, and to provide reference for the local prevention and control of HIV and other sexually transmitted diseases among FSW. Methods A consecutive cross-sectional study was conducted to collect demographic information, awareness of AIDS prevention knowledge, and high-risk behaviors among FSW in Anhui Province from 2010 to 2024. Blood samples were also collected for HIV, syphilis, and HCV antibody testing. The relevant data of the detected objects were statistically analyzed using R 4.5.3 and Joinpoint 5.3.0 software. Results A total of 102 015 FSW were investigated from 2010 to 2024. The overall awareness rate of AIDS prevention knowledge was 88.21%, and the consistent condom use rate during commercial sex in the past month was 63.75%. The positivity rates of HIV, syphilis, and HCV antibodies were 0.06%, 2.15%, and 0.29%, respectively. A decreasing trend was observed for both syphilis (AAPC=-7.43%, 95%CI:-11.02% - -3.70%, t=-4.224，P=0.001) and HCV (AAPC=-5.62%, 95%CI:-9.06% - -2.05%, t=-3.363, P=0.005) antibody positive rates. Multivariate logistic regression analysis showed that aged 30-<50 years, education level of primary school or below, non-local household registration, working in low-tier venues, inconsistent condom use, and lack of HIV prevention knowledge were factors associated with HIV/syphilis infection. Aged ≥50 years, divorced or widowed, non-local household registration, and working in low-tier venues were factors associated with HCV infection. Conclusion From 2010 to 2024, the infection rates of HIV and HCV among FSW in Anhui Province remained at relatively low levels, and the syphilis antibody positive rate showed a declining trend. However, it is still necessary to strengthen health education and behavioral interventions to improve awareness of prevention and testing among this population.