Abstract: An isobaric stable isotope-containing phosphorylated protein labeling reagent, and a preparation method and application thereof are provided. The protein labeling reagent is a phosphorylated dipeptide organophosphorus reagent labeled by a stable isotope such as deuterium-2, carbon-13, oxygen-18, or nitrogen-15. The preparation method includes: (1) preparation of an isobaric stable isotope-containing amino acid with N-terminal protection, (2) preparation of an isobaric stable isotope-containing amino acid activated ester Fmoc/Boc-R1-NHS with N-terminal protection, (3) preparation of an isobaric stable isotope-containing dipeptide, (4) preparation of an isobaric stable isotope-containing phosphite, (5) preparation of an isobaric stable isotope-containing phosphite dipeptide, and (6) preparation of a stable isotope-labeled N-phosphorylated amino acid activated ester.