Gastric cancer (GC) is one of the leading causes of cancer-related death worldwide, largely because of difficulties in early diagnosis. Despite accumulating evidence indicating that aberrant glycosylation is associated with GC, the development of diagnostic platform to capture changes in glycosylation to increase specificity and sensitivity for clinical use is still an analytical challenge. Haptoglobin is the major serum components and a positive acute-phase protein with immunomodulatory properties. Here, we created an analytical platform with a targeted glycoproteomic approach for GC biomarker discovery. Glycosylation alteration was monitored by intact analysis of Hp using liquid chromatography–mass spectrometry followed by immunoaffinity purification with the serum samples. Unlike conventional glycomic approach with untargeted mass spectrometric profiling of released glycan, our platform based on direct analysis of intact glycoprotein has merits such as simple, easy handling of sample preparation and time saving for analysis. Age- and sex-matched 200 serum samples (100 cancer patients and 100 healthy controls) were used to explore the clinically altered glycosylation for gastric cancer diagnosis. This study suggested that glycosylation variation of serum haptoglobin were associated with patients with gastric cancer and might be a promising marker for the cancer screening. Our platform, which provides biological information as well as high sensitivity and reproducibility, may be useful for GC biomarker discovery.