Abstract: In order to rapidly detect in vitro inhibitory effect of probiotics against Gram-positive bacteria（Staphylococcus aureus） and Gram-negative bacteria（Escherichia coli）, in the experiment, the in vitro antibacterial method was used to colonize successively or simultaneously at intervals of 24 h; the competitive exclusion effect of 17 common probiotics such as Lactobacillus and Bacillus isolated from different samples on common pathogenic bacteria（Escherichia coli, Staphylococcus aureus） were observed respectively. According to the established quantitative standard for judging the antibacterial effect of probiotics on pathogenic bacteria, the higher the score, the stronger the mutual inhibitory activity was. The results showed that when probiotics were colonized in the early stage, except for Bacillus vegicus GXNN20201206-5, which had no inhibitory effect on the growth of the two pathogens, the other 16 probiotics had a certain inhibitory effect on the growth of the two pathogens. When the pathogenic bacteria colonized in the early stage, the pathogenic bacteria only had inhibitory effect on four strains of probiotics. When probiotics and pathogenic bacteria were colonized at the same time, Lactobacillus acidophilus GX20200417-2, Enterococcus faecalis GX20200417-3, Lactobacillus plantarum GX20200417-4, and Lactobacillus paracasei GX20200417-5 had the best inhibitory effect on Escherichia coli; Lactobacillus plantarum GX20200417-4 had the best inhibitory effect on Staphylococcus aureus. The results suggested that the inhibitory effect of probiotics on pathogenic bacteria was not only strain-specific, but also related to the time sequence of colonization.