The present invention relates to a method of traceless labeling glycoproteins on a surface and an application thereof. A test glycoprotein can be immobilized on a surface using a BA-tosyl probe and the BA-tosyl probe is then released using a releasing agent, so as to expose a glycan residue of the test glycoprotein. The exposed glycan residue of the test glycoprotein can be detected without altering native glycan structures. Moreover, the present invention further provides a detection kit of traceless labeling glycoproteins on a surface in the study of glycoprotein-protein interactions, which is suitable for using the aforementioned method.