Amyotrophic lateral sclerosis (ALS), characterized by its insidious onset and progressive deterioration, poses a significant diagnostic challenge due to the lack of biomarkers in clinical practice. This study aims to unravel the serum biomarkers associated with ALS. We conducted a proteomic analysis of serum samples from a training group comprising 45 ALS patients and 45 healthy controls using magnetic bead-based immobilized metal ion affinity chromatography (MB-IMAC) and MALDI-TOF-MS technology. The differentially expressed peaks were screened using ClinProTools software and subsequently identified by LC-MS/MS.