Human milk oligosaccharides (HMOs) are essential for babies' growth. The most commonly used method for HMOs analysis combines hydrophilic interaction liquid chromatography (HILIC) with ultra-performance liquid chromatography (UPLC). However, high-pressure operation and the cost of commercial HILIC columns limit progress in the HMOs research. In this study, we synthesized a novel mixed-mode adsorbent, PS-g-PAMPS, by grafting 2-acrylamido-2-methylpropane sulfonic acid (AMPS) onto a Merrifield resin (PS-g-Cl), and characterized it. The adsorbents were packed into a column (Mixed-HILIC column) for efficient high-performance liquid chromatography (HPLC) analysis of four HMOs: 2'-Focusllactose (2'-FL), Lacto-N-Tetraose (LNnT), 3'-Sialyllactose (3'-SL), and 6'-Sialyllactose (6'-SL). Compared to three commercial columns, AMINEX HPX-87H column, ROA-Organic Acid column, Glycan BEH amide column, our column offered several advantages: low-pressure operation, cost-effective adsorbent, and baseline separation of six analytes in the same analysis time. The mixed-mode mechanism of electrostatic repulsion and hydrophilic interactions was verified using zeta potential, and organic phase ratio. Key performance parameters, including spike recovery, limit of detection, and limit of quantification, were evaluated, and the column's reliability was confirmed using a real 2'-FL fermentation sample. These results demonstrated the potential of the Mixed-HILIC column for the development of application chromatography in HMOs.