Lysine acetylation is a dynamic and reversible modification, which has been proved to be a key posttranslational modification in cellular regulation. However, the low amounts of the acetylated proteins could hardly be detected before enrichment. In this study, for the first time, antibody-immobilized magnetic carbonaceous microspheres were developed for selective enrichment of acetylated proteins and peptides. At first, standard proteins composed of acetylated bovine serum albumin, myoglobin, α-casein and ovalbumin were used as model proteins to verify the enrichment efficiency. Then, the synthesized peptide was employed to confirm the selectivity of the method. Besides, the antibody-immobilized magnetic particles were successfully applied to analyze mouse mitochondrial proteins. After database search, 29 acetylated sites in 26 proteins were identi?ed.