HIV-indeterminate Western blotting (WB) results are typically obtained in WB confirmatory assays, and the number of indeterminate samples may increase with the detection of HIV infections, which will present considerable challenges for the management of HIV/AIDS. Nucleic acid detection has been used as a laboratory test for screening suspected or indeterminate samples. However, the effectiveness of these assays for the differential diagnosis of HIV-indeterminate WB samples remained undetermined. In this study, 210 subjects with HIV-indeterminate WB results were detected from 6360 positive HIV screening samples between 2015 and 2016 in southeastern China, in which HIV-indeterminate WB results accounted for 3.30%. The highest proportion of indeterminate results was observed in pregnant and lying-in women receiving physical examinations (16.67%), followed by that in voluntary blood donors (8.82%). The most common WB band patterns were p24, gp160 and p24, and gp160. The follow-up study revealed that the highest negative and positive conversion rates of HIV antibodies were in samples with a single p24 band (80.28%), and with gp160 and p24 bands (86.21%), respectively. Among the Env, Gag, and Pol antibodies, samples with a Gag band showed the highest negative conversion rate (81.25%), whereas the highest positive conversion rate was observed in samples with an Env band (56.76%). In addition, quantitative and qualitative HIV nucleic acid testing exhibited the highest sensitivity (96.3%) and specificity (97.85%), respectively. Our results indicate a lower proportion of HIV indeterminate WB results in southeastern China compared to previous reports, and the follow-up re-examination of patients with HIV indeterminate results should be performed. Nucleic acid testing facilitates the identification of HIV infections.