Based on the Shigella ipaH gene, the RNA-DNA primers and Blockers were designed and synthetized, and the real-time fluorescence single primer isothermal amplification (real-time fluorescence SPIA) for the detection of Shigella was established. The reaction time of the method was 44min. After detection of the 4 different Shigella group strains and 12 other food borne bacteria by the real-time fluorescent SPIA, the results showed that only the Shigella could be detected and showed the typical fluorescence curve. Further studies show that the sensitivity of the detection method for Shigella flexneri in pure culture was 1.16fg/μL and 1.3CFU/mL. The detection limit of Shigella flexneri in milk was 1.8CFU/mL. The results demonstrate that the real-time fluorescence SPIA detection method for Shigella is highly sensitive, strong specific and much more convenient.