ABSTRACT BACKGROUND: Female Sex Workers (FSWs) are a high-risk population for Human Immune Virus (HIV) infection. However, HIV related services for sex workers are given little attention in developing nations like Ethiopia. Thus, this study attempted to assess HIV preventive practice and associated factors among FSWs in the Afar region. METHODS: Community-based cross-sectional study was conducted on 419 FSWs from April 1st to May 2nd in 2018 in four hotspot towns (i.e. Logia, Mille, Gedamayitu and Awash) found in Afar region. The study participants were selected using snowball sampling technique. The sociodemographic and behavioral data were collected using face-to-face interview. Logistic regression analyses were done to determine factors associated with HIV prevention practices. The statistically significant level was declared at a P-value < 0.05. RESULTS: The HIV/AIDS preventive practice among FSWs was 61.1% [95% CI 56.2% - 65.9%]. The multivariable logistic regression analysis showed that being single [AOR=2.57: 95% CI 1.06, 6.21: P<0.05] and urban residence before being FSWs [AOR= 3.26: 95% CI 1.25, 8.54: P<0.05] were found to be enhancing factors of the preventive practice of HIV/AIDS. However, being illiterate [AOR=0.133: 95% CI 0.032, 0.557: P<0.01], young age [AOR=0.065: 95% CI 0.021, 0.196: P<0.001], being alcohol drinker [AOR= 0.104: 95% CI 0.026, 0.417: P<0.01], being cigarette smoker [AOR= 0.096: 95% CI 0.022, 0.414: P<0.01] and lack of aware of HIV transmission [AOR= 0.08: 95% CI 0.03, 0.22: P<0.001] were the hindering factors of the preventive practice of HIV among FSWs. CONCLUSION: The HIV/AIDS preventive practice among FSWs in Afar region hotspot towns (61.1%) is still very low compared to the country progress report on HIV response for FSWs (98%). The regional health bureau should design inclusive strategies to improve the HIV preventive practice among FSWs. These includes regular HIV testing campaigns, sustainable condom distribution in the hotspot towns. The region should encourage NGOs to work on inclusive HIV prevention upon FSWs. Female sex workers should be aware of the risky behaviors that lead to HIV. Additional qualitative methods are also recommended. KEYWORDS: HIV/AIDS, preventive practice, associated factors, female sex workers, Afar region, Ethiopia