The invention provides red fluorescent carbon quantum dots used for detecting pH in cells and a preparation method and application thereof. The preparation method comprises the following steps: (1) weighing 0.1-0.2mmol of p-phenylenediamine solution and dissolving in 0.075-0.15mol of ethylenediamine, to obtain faint yellow liquid; (2) arranging the solution to a microwave oven, reacting for 5-25min with the power of 20-100%, cooling to room temperature after reaction is ended, and adding 5mol/L of HCL until gas escapes; and (3) putting the obtained solution into a dialysis bag of 500-1000Da, then putting the dialysis bag into a containing with water for dialysis for two days, to obtain a pure carbon quantum dot water solution, and performing freeze drying on the obtained pure carbon quantum dot water solution to obtain light pink flocculent carbon quantum dots. The carbon quantum dots prepared according to the invention have the characteristics of long launching, high quantum yield, good biocompatibility and the like; and the carbon quantum dots have sensitive response to pH and can penetrate a nuclear membrane to enter a cell nucleus, so as to enter the whole cell, thus achievingan effect of detecting the pH of the whole cell.