Abstract: The jejunum contents of 5 55-week-old healthy adult scatter-fed laying chicken were taken as samples to dilute to 10-6 with sterile normal saline. 0.1 mL bacteria solution was dropped on MRS flat and cultivated in 37 ℃ incubator for 24 h. The Lactobacillus was separated. The separated and purified Lactobacillus was identified by Gram staining, observation of colonial morphology and sugar fermentation test. The results showed that 20 strains of Lactobacillus were separated and screened out from the jejunum of 55-week-old healthy adult scatter-fed laying chicken. The results of microscopic examination after Gram staining showed that the isolated strains were rod-shaped or round-shaped. 7 species of Lactobacillus was identified by sugar fermentation test, including Lactobacillus salivarius subsp. salivarius, Lactobacillus coryniformis subsp. torquens, Lactobacillus salivarius subsp. salicinius, Lactobacillus agilis, Lactobacillus gasseri, Lactobacillus acidophilus and Lactobacillus delbrueckii subsp. bulgaricus.