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Objective The aim of the present study was to evaluate the performance of the simultaneous detection of HIV-1 RNA, HIV-1 DNA, and HCV RNA using one dried blood spot (DBS) as an alternative sample to plasma. Method A total of 571 paired DBS/plasma samples were collected from men who have sex with men (MSM) and injection drug users (IDUs), and serological and molecular assays were performed. Using plasma results as the reference standard, the performance of DBS tests for HIV-1 RNA, HIV-1 DNA, and HCV RNA was evaluated. Pearson's correlation coefficients and Bland-Altman analysis were performed to assess the correlation and concordance between DBS and plasma. Results Among paired plasma/DBS samples with detectable HIV-1 RNA and HCV RNA, five samples (5/32) were not detectable in DBS, while measurable HIV-1 RNA levels were present in plasma (1.44 to 3.99 log10 copies/mL). There were two samples (2/94) with undetectable HCV RNA in DBS, while measurable HCV RNA levels were present in plasma (?5 to 5.99 log10 copies/mL). The correlation between HIV-1 RNA light chain variable region (VL) values obtained from plasma and DBS showed that r = 0.683 (P < 0.01), n = 27 and r = 0.612 (P < 0.01), n = 89 in HCV RNA. Bland-Altman analysis revealed that in HIV-1 RNA, the mean (± SD) difference between HIV-1 RNA in plasma and DBS was 1.00 ± 1.01 log10 copies/mL, and all samples were within ± 1.96 SD (?0.97 to 2.97 log10 copies/mL) for DBS. The mean difference (± SD) in HCV RNA was 0.15 ± 1.08 log10 copies/mL, and 94.38％ (84/89) were within ± 1.96 SD (?1.96 to 2.67 log10 copies/mL). Overall, HIV-1 RNA and HCV RNA levels obtained from a DBS were lower than those obtained from plasma. HIV-1 DNA in a DBS showed concordant results with HIV-1 RNA in plasma. HIV-1 DNA RT-PCR using a DBS showed acceptable performance. Conclusion The performance of the simultaneous detection of HIV-1 RNA, HIV-1 DNA, and HCV RNA using one DBS was acceptable. DBS, as an alternative sample to plasma, may be a viable option for the simultaneous detection of HIV-1 RNA, HIV-1 DNA, and HCV RNA in resource-limited settings or for individuals living in areas that are difficult to access.