The increased prevalence of metabolic dysfunction-associated steatotic liver disease (MASLD) and its profound implications for global health have sparked extensive research endeavors aimed at developing potential diagnostic methods for this condition. Despite the achievements in defining various environmental factors and genetic predispositions linked to MASLD, diagnosis and clinical staging of the disease remain challenging. Recently, extracellular vesicles (EVs) have garnered considerable attention owing to their roles in metabolic dysfunctions and their potential as biomarkers for various conditions. This study aimed to investigate whether microRNAs (miRNAs) in serum EVs could be utilized for diagnosing and staging MASLD. We applied an innovative and efficient approach that involves capturing and analyzing extracellular vesicles using wheat germ agglutinin (WGA)-coupled magnetic beads, subsequently employing reverse transcription quantitative polymerase chain reaction (RT-qPCR) for analysis. MiR-574-3p, miR-542-3p, and miR-200a-3p in serum extracellular vesicles were significantly elevated in patients with MASLD, indicating their potential as diagnostic markers. This study has established a straightforward assay platform for isolating extracellular vesicles without the need for purification and for quantitatively detecting miR-574-3p, miR-542-3p, and miR-200a-3p in serum extracellular vesicles.