The invention discloses a mitochondrial pH fluorescent probe based on benzothiazole, and a preparation method and an application thereof. The probe is specifically named as 2-(2-(6-hydroxynaphthalene-2-yl)vinyl)-3-(6-(triphenylphosphonyl)hexyl)benzothiazole-3-bromide (HTBT2). The preparation method comprises the steps: firstly, carrying out a reaction of 2-methylbenzothiazole and 1,6-dibromohexaneunder heating conditions to prepare 3-(6-bromohexyl)-2-methylbenzothiazole-3-bromide (BMBI), and then carrying out mixed reflux of the prepared BMBI with triphenylphosphine and acetonitrile to prepare 2-methyl-3-(6-(triphenylphosphorus)hexyl)benzothiazole-3-bromide (MTBI); and finally, dissolving MTBI and 6-hydroxy-2-naphthaldehyde in ethanol, and adding a small amount of piperidine, refluxing, and then separating and purifying to obtain HTBT2. The probe has the pKa value of 8.04+/-0.02 and is quite close to that of a mitochondrial matrix (with the pH of 8.0). At the same time, the probe hasthe advantages of high sensitivity to the changes of the pH, good selectivity and large Stokes displacement, and can be used for monitoring the changes of the pH in cell mitochondria.