BACKGROUND: The availability of oral fluid HIV rapid testing provides an approach that may have the potential to expand HIV testing in China, especially among most-a-risk populations. There are few investigations about the acceptability of oral fluid HIV testing among most-at-risk populations in China. METHOD: A cross-sectional study with men who have sex with men (MSM), female sex workers (FSW) and voluntary counseling and testing (VCT) clients was conducted in three cities of Shandong province, China from 2011 to 2012. Data were collected by face-to-face questionnaire. RESULTS: About 71% of participants were willing to accept the oral fluid HIV rapid testing, and home HIV testing was independently associated with acceptability of the new testing method among MSM, FSW and VCT clients (AOR of 4.46, 3.19 and 5.74, respectively). Independent predictors of oral fluid HIV rapid testing acceptability among MSM were having ever taken an oral fluid HIV rapid test (AOR= 15.25), having ever taken an HIV test (AOR= 2.07), and education level (AOR= 1.74). Engagement in HIV-related risk behaviors (AOR= 1.68) was an independent predictor of acceptability for FSW. Having taken an HIV test (AOR= 2.85) was an independent predictor of acceptability for VCT clients. The primary concern about the oral fluid HIV testing was accuracy. The median price they would pay for the testing ranged from 4.8 to 8.1 U.S. dollars. CONCLUSION: High acceptability of oral fluid HIV rapid testing was shown among most-at-risk populations. Findings provide support for oral rapid HIV testing as another HIV prevention tool, and provide a backdrop for the implementation of HIV home testing in the near future. Appropriate pricing and increased public education through awareness campaigns that address concerns about the accuracy and safety of the oral fluid HIV rapid testing may help increase acceptability and use among most-at-risk populations in China.