"The HIV epidemic in Ghana is characterized as a mix of a low-level generalized epidemic with significant contributions from transmission among female sex workers (FSW) and their clients. This study seeks to identify and describe key characteristics and sexual behaviors of FSW and estimate the prevalence of HIV, syphilis, gonorrhea, chlamydia, and hepatitis B virus (HBV) among FSW in Ghana. Method A total of 7,000 FSW were recruited for the study using Time Location Sampling (TLS) approach with 5,990 (85.6%) participants completing both biological and the behavioral aspects of the study. A structured questionnaire was administered to respondents to assess several factors, such as background characteristics, sexual risk behaviors, condom usage, HIV/AIDS knowledge, opinions, and attitudes. Trained staff conducted face-to-face interviews using mobile data collection software (REDCap) after provision of specimens for HIV and STI testing. Descriptive statistics such as medians, ranges, charts, and percentages are performed and presented. Also included, are bivariate analyses to establish relationships between FSW type and other relevant characteristics of the study. Results Among the 7,000 (100%) FSW sampled from all regions, 6,773 took part in the behavioral and 6,217 the biological. There were 783 (11.2%) respondents who took part only in the behavioral and 227 (3.2%) only in the biological. Most were young, with a median age of 26 years, majority had never been married or were widowed/divorced and a quarter had no education or had only primary education. Majority (74.8%) of FSW first sold sex at age 25 years or less with a median age of 20 years. Most (84.8%) of the FSW indicated that they entered sex work for money, either for self or family and had an average of eleven (11) sexual partners per week. More than half (55.2%) of the FSW were new entrants who had been in sex work for less than 5 years before the study. Consistent condom use with paying clients was generally unsatisfactory (71%), and was however, very low (24%) with their intimate partners or boyfriends. Only about half (54.6%) of FSW have been exposed to HIV prevention services in the last three months preceding the survey, and this varies across regions. Overall, comprehensive knowledge about HIV and AIDS was low. Only 35% of FSW had comprehensive knowledge. HIV prevalence was 4.6% and was higher among seaters (brothel-based) and older FSW who had been sex work for a longer period. The HIV prevalence from the previous bio-behavioral survey (BBS) in 2015 and 2011 were estimated to be 6.9 and 11.1%, respectively. Conclusion Compared to the results from the previous studies, the findings give an indication that Ghana is making significant progress in reducing the burden of HIV among FSW in the country. However, risky behaviors such as low consistent condom use, low coverage of HIV services across the regions, and low comprehensive knowledge could reverse the gains made so far. Immediate actions should be taken to expand coverage of HIV services to all locations. Efforts must be made to reach out to the new entrants while also addressing strongly held myths and misconceptions about HIV"