Provided are an enzyme treatment apparatus for proteins using a hollow fiber membrane and an on-line proteomics method using same. The enzyme treatment apparatus for proteins according to the present invention can increase a recovery rate of peptides, which are recovered through an enzyme treatment process, by basically solving the problem of low reproducibility of an enzyme activity which may occur during a conventional enzyme treatment process, and can also reduce a time for purification and provide higher yield by performing separation and purification through a single step. In particular, the present invention can be usefully applied for developing a disease-specific protein biomarker through a statistical analysis method having highly efficient detectability for proteins in research for finding biomarkers related to human diseases.