Duck viral hepatitis (DVH) mainly affects ducklings under 1 month of age, causes liver necrosis, enlargement, and hemorrhage, and is highly lethal, seriously jeopardizing the duck industry. The prevalence of serotypes DHAV-1 and DAstV-3 is increasing, and co-infection is common. Moreover, the similar clinical characteristics of the DHAV-1 and DAstV-3 infections and high frequency of co-infection make diagnosis difficult. In this study, to establish a method for the rapid simultaneous detection of DHAV-1 and DAstV-3 , two pairs of specific primers were designed according to their conserved gene regions. A SYBR Green I-based real-time PCR assay was successfully established that can quickly and differentially detect the two viruses. Moreover, the assay is highly specific and does not show cross-reaction with other common viruses. The detection limit of the method is 7.34 × 10 1 copies/μL and 3.78 × 10 1 copies/μL for DHAV-1 and DAstV-3, respectively, indicating high sensitivity. A total of 34 clinical samples were tested using the established method; the positive rates for DHAV-1 and DAstV-3 was 14.71% and 8.82%, respectively, and that for co-infection was 2.94% (1/34), which was better than that obtained with conventional PCR. In summary, the SYBR Green I-based real-time PCR assay established in this study has high specificity, good sensitivity and accuracy, high feasibility, and is rapid. Thus, it can be a powerful tool for the co-infection detection of DHAV-1 and DAstV-3 and for future epidemiological studies.