The invention discloses a method for preparing pH-responsive carbon nanotube fluorescent probe in the technical field of nano materials, which comprises the following steps: mixing a carbon nanotube raw material and an oxidizing acid, sequentially subjecting the mixture to supersonic treatment, stirring treatment and suction filtration, and washing the product of the suction filtration till the product is neutral to obtain purified carbon nanotubes; mixing the purified carbon nanotubes with the oxidizing acid again, sequentially subjecting the mixture to supersonic treatment, secondary stirring treatment and suction filtration, and washing the product of the suction filtration till the product is neutral to obtain purified cut nanotubes; and subjecting aqueous solution of purified cut nanotubes to supersonic treatment, adding acridine orange into the aqueous solution, subjecting the mixture to supersonic treatment again, keeping the mixture away from light, stirring the mixture, subjecting the mixture to suction filtration, washing and dialyzing the product of the suction filtration, and drying the product at room temperature under vacuum to obtain the pH-responsive carbon nanotube fluorescent probe. The preparation method provided by the invention is simple, easy to implement and high in controllability. The product of the method has a unique pH- responsive property and has wide application prospects in terms of nanoscience, materials science, biomedical science and the like.