Lipid was automatically coupled to DNA chains with i motif structure marked Cy3 and Cy5 at both ends by DNA synthesizer to form a DNA-lipid probe. The probe could be directly inserted into the surface of cell membrane by strong hydrophobic interaction between lipid and cell membranes, and realized the ratio detection of extracellular pH value under hypoxic and normoxic conditions. Under the condition of high pH, the fluorescent groups at both ends of the DNA strand with i-motif structure were separated without any FRET effect, while in the condition of low pH, the DNA strand with i-motif structure formed tetramer structure on the cell surface, and the fluorescent groups at both ends were close to each other, resulting in strong FRET effect.Quantitative detection of pH could be achieved by measuring the fluorescence intensity ratio of the two fluorophores.Accurate detection on extracellular pH in hypoxic environment using this probe is of great significance in physiology and pathology.