Background Due to the unavailability of an effective vaccine or antiviral drug against the African swine fever virus (ASFV), rapid diagnosis methods are needed to prevent highly contagious African swine fever. Objectives The objective of this study was to establish the ladder-shape melting temperature isothermal amplification (LMTIA) assay for the detection of ASFV. Methods LMTIA primers were designed with the p72 gene of ASFV as the target, and plasmid pUC57 was used to clone the gene. The LMTIA reaction system was optimized with the plasmid as the positive control, and the performance of the LMTIA assay was compared with that of the commercial real-time polymerase chain reaction (PCR) kit in terms of sensitivity and detection rate using 200 serum samples. Results Our results showed that the LMTIA assay could detect the 10 4 dilution of DNA extracted from the positive reference serum sample, which was the same as that of the commercial real-time PCR kit. The coincidence rate between the two assays was 100%. Conclusions The LMTIA assay had high sensitivity, good detection, and simple operation. Thus, it is suitable for facilitating preliminary and cost-effective surveillance for the prevention and control of ASFV.