Highly site-selective dimethyl labeling of N-terminus of peptides has been obtained by adjusting the acidic strength of the reaction solution. And this selective labeling strategy combined with the use of different isotope formaldehyde reagents has been successfully used in the proteome quantification by using the isobaric peptide cross-sequence labeling method, which would play increasingly important roles in the clinical diagnosis, especially in the discovery of biomarkers for diseases.