Small extracellular vesicles (sEVs) derived from gastric juice (GJ) have emerged as potential biomarkers for gastric disease. However, methods for isolating sEVs from GJ (gsEVs) remain underexplored.This study employed four methods for isolating gsEVs: ultracentrifugation (UC), PEG6000 precipitation combined with UC (PEG-UC), UC combined with size exclusion chromatography (UC-SEC), and ultrafiltration combined with SEC (UF-SEC). The yield and purity of gsEVs isolated by each method were evaluated, and the proteomic profile of gsEVs was examined through label-free quantitative proteomics. Additionally, a series of validation experiments were conducted to identify specific biomarkers for gsEVs.The results revealed that gsEVs isolated using the UC method exhibited the highest purity and the largest number of identified proteins compared to the other methods. Notably, the gastric tissue-specific peptide trefoil factor 2 (TFF2) was highly expressed in gsEVs isolated by all methods, suggesting that TFF2 may serve as a specific biomarker for gsEVs. Validation experiments showed that TFF2 was exclusively present in gsEVs and not in the GJ supernatant after UC. Furthermore, TFF2 was selectively expressed in gsEVs, but not in sEVs derived from other biofluids such as intestinal juice and plasma. The efficiency of TFF2 as a gsEVs biomarker was higher than the commonly used biomarker CD9, CD81, and Syntenin-1. Multi-omics analysis indicated that the functions of gsEVs carrying TFF2 were primarily associated with inflammation and cancer.The UC method is suitable for isolating gsEVs, particularly for mass spectrometry-based proteomic analysis. The small peptide TFF2 may serve as a potential specific biomarker for gsEVs. This study offers new insights for research on sEVs in gastric diseases.© 2025 Fan et al.