Pre-exposure prophylaxis (PrEP) is an effective strategy to prevent uninfected individuals from contracting human immunodeficiency virus (HIV), however it must be acceptable to stakeholders in order to be effective. This study aimed to assess the acceptability of PrEP and related influencing factors. A cross-sectional survey was conducted among female sex workers (FSW), people who inject drugs (PWID), and men who have sex with men (MSM) using respondent driven sampling. Factors influencing PrEP acceptability were estimated using ordinal logistic regression and Bayesian networks. The survey included 765 eligible participants. The mean score of the perceived acceptability index was 3.9 (SD = 1.97). Multivariable logistic regression analysis revealed a higher acceptance of PrEP was associated with elder age, having other medical insurance, higher perceived utility of PrEP in facilitating prevention of HIV, higher perceived ease of use, higher perceived risk of increased risk behavior, higher perceived privacy problem in using PrEP, higher perceived comparative advantage over condom use, higher perceived comparative advantage of having sex when the urge arises, and higher perceived image of PrEP user as having sexual risky behavior, as public-minded and as health-conscious. The Bayesian network model showed perceived ease of use, perceived image of user as health-conscious, and perceived comparative advantage of having sex when the urge arises were directly associated with acceptability of PrEP. If these three factors were at a high level, 74.6% of the participants would have a high level of acceptability of PrEP. Effective education strategies to promote the acceptance of PrEP are needed. Implementation strategies should incorporate more inclusive messaging and build positive publicity for PrEP to reduce the stigma that PrEP use indicates risky behavior.