African swine fever (ASF) continues to cause outbreaks throughout regions of Africa, Europe and Asia. The disease can cause severe morbidity and mortality resulting in serious economic losses. Since there is no vaccine available to control ASF, early detection is critical to contain and control the disease. The aim of this study was to develop a novel real-time PCR assay based on highly conserved ASFV gene E183L (p54). The limit of detection of the assay, VNUA-54 real-time PCR, was 2.63 copies/reaction and 2 Log10 HAD50/ml. The VNUA-54 real-time PCR was able to detect fifteen different ASFV reference strains representing p72 genotypes I, II and V. The assay was specific and did not amplify other swine viruses including CSF, FMD, PRRS, and PED. The diagnostic sensitivity of the real-time PCR assay was evaluated using 187 field clinical specimens collected from swine farms located in different provinces in Vietnam. The VNUA-54 real-time PCR assay is an additional tool for ASF diagnostics and can be used in combination with other p72 based ASFV real-time PCR assays as a rapid confirmatory assay.