African swine fever virus (ASFV) is the pathogen of African swine fever, a highly contagious and fatal disease of wild boar and domestic pigs. The flow of ASFV through pork products is more concealed, higher risky, and more difficult to prevent and control. Presently, on-site ASFV detection methods in preclinical infected pigs and circulated pork products are lacking. Here, fluorescent test strip-based rapid ASFV detection method in pork was established combined with recombinase aided amplification (RAA) and quantum dot microspheres (QDMs). This method is specific to ASFV with no cross-reactivity to pseudorabies virus (PRV), porcine circovirus type 2 (PCV2), and porcine reproductive and respiratory syndrome virus (PRRSV). The method also showed highly sensitivity with a detection limit of 1 copy for ASFV plasmid templates containing B646L gene and 100 copies/g for DNA extracts from clinical pork samples within a short detection time of less than 25 min. Additionally, the method showed 99.17% consistency with real-time PCR in the ASFV detection of 120 clinical pork samples. Overall, the QDMs-based test strip method provides specific, sensitive, rapid, and simple detection of ASFV in pork, which may contribute to maintain the food safety of pork products, and facilitate ASFV traceability and prevention. Rapid and sensitive detection of African swine fever virus in pork by QDMs based test strip assay.