The invention relates to a hydrophobic group modification-based protein N-terminal enrichment method, which comprises the steps of sealing free amino groups in protein level, carrying out marking through a strong hydrophobic group, and carrying out affiliative trapping through a reverse phase material. Particularly, the method comprises the steps of firstly sealing free amino groups of an N terminal and a side chain in protein level by adopting chemical modification and carrying out enzymolysis on the protein; introducing the strong hydrophobic group to a non-N-terminal peptide segment through newly generated free amino groups; and finally removing the non-N-terminal peptide segment with the strong hydrophobic group by adopting the reverse phase material in an affiliative manner to obtain an N-terminal peptide segment of the protein. The hydrophobic group modification-based protein N-terminal enrichment method has the advantages of being high in mark efficiency, high in removal efficiency, high in selectivity and simple in processing step, and the sample loss and nonspecific adsorption are reduced.