African swine fever virus (ASFV), the aetiological agent of African swine fever (ASF), causes lethal haemorrhagic fever in domestic pigs with high mortality and morbidity and has devastating consequences on the global swine industry. On-site rapid and sensitive detection of ASFV is key to the timely implementation of control. In this study, we developed a rapid, sensitive and instrument-free ASFV detection method based on CRISPR/Cas12a technology and lateral flow detection (named CRISPR/Cas12a-LFD). The limit of detection of CRISPR/Cas12a-LFD is 20 copies of ASFV genomic DNA per reaction, and the detection process can be completed in an hour. The assay showed no cross-reactivity with other swine DNA viruses, and has 100% agreement with real-time PCR detection of ASFV in 149 clinical samples. Overall, the CRISPR/Cas12a-LFD method provides a novel alternative for the portable, simple, sensitive, and specific detection of ASFV and may contribute to the prevention and control of ASF outbreaks.