The outbreak of African swine fever (ASF) in China in 2018, caused a huge economic loss on the pig industry. To accurately diagnose clinical infection with ASF virus (ASFV), we developed a duplex TaqMan-probe-based real-time PCR for the simultaneous detection of two discontinuous genes in the virus genome to avoid false-negative in the clinical diagnosis. Two sets of ASFV-gene specific primers, along with two TaqMan fluorescent probes were designed to target the conserved region of B646L and B438L genes, respectively. The method showed high sensitivity and specificity, with a detection limit of 10 copies/μl without cross-reactions with other pathogen genomes of CSFV, PRRSV, PEDV, PRV, PPV and PCV2. 269 clinical swine samples were tested using this method, and the results of 60 samples showed excellent consistency (95.0%) with a commercial kit. This method will greatly improve the efficiency for surveillance of ASFV and can help reduce economic losses to the swine industry, which also benefits animal and public health.