Objective To study the feasibility of using dried blood spot (DBS) specimens in BED-CEIA assay and to estimate the HIV-1 incidence with DBS in China. Methods 10 313 participants from 27 voluntary counseling and testing(VCT) sites were enrolled in this study. Both plasma and DBS specimens were collected. 349 eligible pairs of HIV-1 positive specimens confirmed by Western Blot were available for BED-CEIA and the results compared. Negative control, low positive control, high positive control and calibrator in DBS were used to evaluate the stability and repeatability of the detection results of DBS specimens. Results Of the total HIV positives, 294 and 53 participants were tested as long-term or recent HIV infections by both DBS and plasma specimens. Two participants showed different results from DBS and plasma specimens, but their final An values were close to the cutoff value of BED-CEIA assay. The concordance rate between the two types of specimens was 99.43% and the R2 linear of the stability and repeatability results of DBS specimens reached 0.9551 and 0.95, respectively. Conclusion Although some individual specimens showed different results, the incidence rates calculated from both types of specimen were equivalent. DBS specimens were suitable for estimating the HIV incidence and could be popularized in China. However, further studies regarding window period and adjustment coefficients of BED-CEIA assay are needed.