Foot-and-mouth disease (FMD) is an acute and highly contagious animal disease that is caused by FMD virus (FMDV), and it brings huge economical damage to the livestock industry once breaking out. Vaccination with inactivated FMDV is widely used to prevent FMD prevalence, but there are several limitations to the conventional inactivated vaccine such as virus escape and high production costs. To avoid these deficiencies, recombinant protein subunit vaccines are being developed for the prevention and control of FMD. The structural protein VP1 of FMDV contains most of the pivotal immune epitopes, therefore numerous researchers focus on VP1 for diagnosis and vaccines development for FMDV. Compared with the eukaryotic expression system, the prokaryotic expression system has the advantages of fast growth, easy scaling up and low cost, making it more suitable for animal vaccine production. However, VP1 tends to form insoluble and inactive inclusion bodies when expressed in the prokaryotic system. In this study, a strategy for soluble expression of FMDV VP1 (serotype O/MYA/98) in the prokaryotic system was firstly designed by fusion with two tags of small ubiquitin-related modifier (SUMO) and RNA-interacting domain (RID). Through the PCR (polymerase chain reaction) overlap technique, the gene of RID-SUMO-VP1 was constructed and cloned into pET28a(+) vector. Then the recombinant plasmid was transformed into E. coli BL21(DE3). After induction with 1 mmol/L IPTG for 4 h at 18℃, the fusion protein of RID-SUMO-VP1 was efficiently expressed up to 79% insoluble form, while VP1 fused with one tag of SUMO or RID was still expressed as inclusion bodies. After cell disruption, a simple method of two-step ammonium sulfate precipitation was developed, which could separate RID-SUMO-VP1 from the lysate with a purity of 93%. ELISA (enzyme linked immunosorbent assay) showed that RID-SUMO-VP1 could react with O-type FMDV positive mice serum. These results demonstrated that soluble and immunogenic FMDV VP1 can be expressed in E. coli by fusion with a double-tag of RID and SUMO. Our study lays the foundation for the development of immunodiagnostic and novel subunit vaccines of FMD.