[Objective] To structure a type A foot-and-mouth disease (FMD) marker virus with deletion of 104-115 amino acids of the nonstructural protein 3A, then to analyze its biological characteristics and the potential of developing marker vaccine.[Methods] Using overlap extension PCR method, we introduced the deletion of 104-115 amino acids of the nonstructural protein 3A into FMDV A/Sea-97/CHA/2014 full-length infectious clone pQAHN to generate a recombinant full-length plasmid. The marker virus was rescued after transfecion linearized recombinant plasmid into BSR/T7 cells expressing T7 RNA polymerase and identified by RT-PCR, sequencing analysis, indirect immunofluorescence and Western blotting. The plaque and one-step growth curves were used to analyze the biological characteristics of the marker virus. A developed block ELISA method targeting to the deleted epitope of 3A was used to analyze the potential of differentiating animals infected with the marker virus and the wild type virus.[Results] We rescued a type A FMD marker virus containing deletion of 104-115 amino acids of 3A protein. The deletion did not affect the plaque phenotype and one-step growth curve of the marker virus. A developed block ELISA method targeted to the deleted epitope could clearly differentiate animals infected with the marker virus and the wild type virus.[Conclusion] The marker virus containing deletion of the dominant epitope of 3A is suitable as a vaccine candidate strain of differentiating infected from vaccinated animals to effectively prevent and control type A FMD in future.