Changes in the glycosylation content, especially in specific proteins, are of great importance for interpreting the mechanisms and development of certain diseases. However, current detection techniques are limited by the weak ionization efficiency of glycosyls and poor anti-interference of fluorescence signals. Herein, we present a general <i>in situ</i> quantification strategy for protein-specific glycoforms by constructing a recyclable DNAzyme motor for mass spectrometric detection using MUC1-specific sialic acid (Sia) as a model. This approach relies on a DNAzyme-based recycling strategy and two well-designed probes: a protein and a glycan probe. The protein probe consists of an aptamer and a DNAzyme. The glycan probe contains three functional domains: a DNAzyme complementary sequence, a substrate peptide segment, and a dibenzocyclooctyne tag. First, these two probes bind to their corresponding targets and trigger hybridization between adjacent probes on the same protein. With the help of the metal cofactor, the DNAzyme of the protein probe hydrolyzes the double-stranded glycan probe. The protein probe then reverts to a single-stranded state and remains intact for the next round of hybridization and cleavage. In this way, the recyclable DNAzyme motor can hydrolyze all glycan probes bound to the target protein. Finally, the reporter peptide released from the hydrolyzed glycan probes can be quantified by mass spectrometry, thereby converting the signal of the protein-specific glycoform to that of mass spectrometry. This strategy has been successfully used for <i>in situ</i> quantification of MUC1-specific Sia in different breast cancer cell lines. It provides a promising platform for protein-specific glycoform quantification.