The invention discloses an imidazo[1,2-a]pyridine near-infrared ratio type pH fluorescence probe, a preparation method of the probe and application of the probe in measuring the extremely-acid pH change in living cells and escherichia coli. Under inert gas protection, 4-dimethylamino-cinnamaldehyde or a derivative thereof, 4-chloroaniline and sodium bicarbonate are dissolved in methyl alcohol, andafter a heating reflux reaction is conducted, a reaction product and 2-(4-methoxyphenyl)-7-methyl imidazo[1,2-a]pyridine are dissolved in N,N-dimethylformamide; potassium tert-butoxide is taken as analkaline reagent, and a heating reflux reaction is conducted; after concentration, silicagel column separation is conducted. Under the extremely acid condition, a transmitted wave of the probe is longer than 600 nanometers, the transmitted wave is located in a near infrared region, a ratio transmission fluorescence characteristic is presented, and the probe has the advantages of high sensitivityand good selectivity for H+, large Stokes displacement and the like.