Human herpesvirus 8 (HHV-8), also known as Kaposi's sarcoma (KS)-associated herpesvirus (KSHV), is not routinely isolated in cell cultures, and thus detection of HHV-8-specific antibodies is usually performed. In this study, we performed recombinant antigens ORF66- and ORFK12-based Western blot strip assays and ELISA, and surveyed the seroprevalence of HHV-8 antibodies in HIV-positive and -negative patients. In serum samples from patients with positive plasma HHV-8 DNA, the sensitivity of the Western blot strip assay was 100% for the anti-ORF66 antibodies and 83.3% for the anti-ORFK12 antibodies. In addition, ORF66-based ELISA showed higher levels of specificity (87.3%) and sensitivity (84.8%) than ORFK12-based ELISA. Moreover, the area under the receiver-operating characteristics curves (AUROC) was 0.76 for ORF66-based ELISA and 0.66 for ORFK12-based ELISA. The seroprevalence of HHV-8 antibodies to ORF66 and/or ORFK12 in the HIV-infected patients (55%, 97/176) was significantly higher than in the DM patients (45%, 135/301) (P = 0.03) and the HIV-/DM-negative group (11%, 11/100) (P < 0.01). In the HIV-infected patients, the seropositivity of the HHV-8-specific antibody was 30% to both antigens, 19% to ORFK12 and 5.7% to ORF66. Importantly, HHV-8 seropositivity in the HIV-infected patients was significantly associated with the transmission method of intravenous injection and high levels of HIV RNA loading (P < 0.01), but not with gender, CD4 cell numbers or AIDS symptoms. This study assessed the sensitivity and specificity of ORF66 and ORFK12 for the detection of HHV-8 antibodies, providing novel antigens for the diagnosis of HHV-8 infection and epidemiology of HHV-8 seroprevalence.