Diabetes has emerged as a significant public health concern. The existing diabetes diagnostic markers, such as fasting glucose and glycated hemoglobin, have limitations in accuracy and sensitivity in epidemiological investigations. This study identified new serum biomarkers for diabetes through a three-step process: screening, validation, and correlation analysis. By excluding proteins that exhibit opposing trends in DDA and DIA results, we identified 170 differentially expressed proteins. Subsequent quantification of six differential proteins in the plasma of 38 diabetic patients and 32 controls through ELISA and immunoturbidimetry showed a significant upregulation of Adipocyte plasma membrane-associated protein (APMAP), Galectin-3-binding protein (LG3BP), Cathepsin D (CATD), and Beta-2-microglobulin (B2MG) in the diabetic group. Notably, LG3BP demonstrated a strong association with established diabetes markers and risk factors in the correlation analysis, particularly waist circumference, suggesting its potential as a serum/plasma biomarker for diabetes closely related to central obesity. Those findings not only contribute to the repertoire of diagnostic biomarkers for diabetes but also provide a robust analytical framework for the identification of specific protein markers in large-scale serum/plasma samples.