Glycosylation-omics has emerged as a prominent field for early detection and diagnosis by identifying alterations in glycosylation patterns linked to cancer. In the realm of clinical multi-glycosylation-omics applications, there is a critical need for robust, efficient, and cost-effective preprocessing methodologies capable of handling large sample cohorts. To bridge this gap, we introduce the GlycoPro platform, an innovative solution designed to overcome the limitations of existing analysis methods. Tailored for multi-glycosylation-omics sample preprocessing, GlycoPro refines existing workflows by seamlessly integrating steps including protein extraction, desalting, digestion, derivatization, and enrichment. The GlycoPro platform employs a 96-well plate format, enabling the efficient enrichment or desalting of up to 384 samples in a single day. This capability represents a significant increase in throughput, meeting the demands of large-scale clinical sample preprocessing for mass spectrometry analysis. The GlycoPro platform was used to successfully enrich serum N-glycans from breast cancer patients, revealing unique glycomic signatures that distinguish malignant from benign conditions. We have developed a robust N-glycan biomarker panel, demonstrating a sensitivity of 88.24% and a specificity of 78.95% in diagnostics.