African swine fever (ASF) is a devastating disease, which is causing huge economic losses in China. Therefore, it is urgent to provide a rapid, highly specific and sensitive diagnostic method for the detection of African swine fever virus (ASFV), the ASF infectious agent. In this study, a novel quantitative real-time polymerase chain reaction (qPCR) assay with lyophilized powder reagents (LPR), targeting the major structural protein p72 gene, was established for the detection of ASFV. This assay had many advantages, such as saving time and money, good sensitivity and repeatability. The sensitivity of this assay was 100 copies/μl of ASFV plasmid templates, and the assay showed 10-fold greater sensitivity than a qPCR assay recommended by OIE. Furthermore, specificity analysis showed that the qPCR with the LPR for ASFV had no cross-reactivity with other important swine pathogens. In clinical diagnoses of 218 blood samples of domestic pigs in China, the positive rate of the diagnosis of ASFV by qPCR with the LPR and commercial kit reached 80.73% (176/218) and 76.61% (167/218), respectively. The coincidence rate between the two assays is 92.20% (201/218), and kappa value is 0.768 (P < 0.0001) by SPSS analysis. The overall agreement between the two assays was 95.87% (209/218). Further Pearson correlation and linear regression analysis showed a significant correlation between the two assays with an R value of 0.9438. The entire procedure, from specimen processing to result reporting, can be completed within 2 hours. Our results demonstrated the qPCR-LPR assay is a good laboratory diagnostic tool for sensitive and efficient detection of ASFV. This article is protected by copyright. All rights reserved.