Conventional treatment for chronic HCV infection relies on the combination of peg-interferon and ribavirin therapy. Both interleukin-28B (IL-28B) polymorphisms and HCV genotypes serve as the strongest predictive values for therapeutic prognosis. The treatment regimens for HIV/HCV co-infected patients are more complex and dependent on various host immune and viral factors. A rapid and cost-effective IL-28B genotyping tool is therefore crucial to assist clinicians on better patient management. This study aimed to evaluate the performance of a newly developed HybProbe duplex real-time PCR assay in detecting IL-28B polymorphisms on rs12979860 and rs8099917, and to estimate the prevalence of IL-28B polymorphisms among HIV/HCV co-infected patients in Hong Kong. A total of 88 HIV/HCV co-infected patients were recruited at the Integrated Treatment Centre during 2009 to 2014. IL- 28B polymorphisms on rs12979860 and rs8099917 were determined by an in-house HybProbe assay with melting curve analysis. For assay evaluation, IL-28B polymorphisms of 46 samples with diverse HIV/HCV genotypes were confirmed by Sanger sequencing. Both in-house HybProbe assay and sequencing results for IL28B polymorphisms determination were completely concordant. Among the 88 HIV/HCV co-infected, the frequency of rs12979860 wildtype (C/C) was 88.6%, heterozygous mutant (C/T) was 9.1% and remaining 2.3% homozygous mutant (T/T). The prevalence of IL-28B polymorphisms in rs8099917 was slightly differed, which had 90.9% wild-type (T/T), 6.8% heterozygous mutant (G/T) and 2.3% homozygous mutant (G/G). This novel duplex assay could allow clinicians to make early decision on treatment option for HIV/HCV co-infected patients by detecting rs12979860 and rs8099917 polymorphisms simultaneously.