Abstract: In order to establish and apply a real-time fluorescence single primer isothermal amplification ( real-time fluorescence SPIA) for Listeria monocytogenes ( L. monocytogenes) , the RNA/DNA primers and Blockers were designed, which based on hly A gene of L.monocytogenes.The real-time fluorescence SPIA was established to detect the L.monocytogenes after the optimization of the reaction system and reaction conditions, which specificity and detection limit were tested. To detect and compare the DNA of different extraction methods.The results showed that the optimum reaction temperature of fluorescence dye SPIA method was 58 ℃ and the specificity of the primers was good in 30 min, only 4 different sources of L.monocytogenes DNA produced a typical S type fluorescence amplification curve. The detection limit of real-time fluorescent SPIA for L.monocytogenes DNA was 3.6 × 10¹fg/u L and the concentration of corresponding bacteria was 1.2 × 10¹CFU/m L in pure culture.The DNA was extracted by four methods, which were kit method, boiling method, lysozyme protease K method, and saturated phenol method were detected by the real-time fluorescence SPIA. There was no significant difference of the four amplification curves Ct value.The detection limit of real-time fluorescent SPIA for L.monocytogenes DNA was 1.1 × 10²CFU/g for DNA from pork ham samples was extracted by boiling method.The results demonstrate that the new real-time fluorescence SPIA method for Listeria monocytogenes, which has high specificity and high sensitivity, is not affected by the purity of DNA.