Among various solid tumors, gastric cancer (GC) is one of the leading causes of cancer-related deaths worldwide. Expansion into peritoneal cavity which results from dissemination of diffuse cancer cells is the main cause of mortality in gastric adenocarcinoma patients. Therefore, investigation of putative biomarkers involved in metastasis is prerequisite for GC management. In an effort to discover potential tumor markers associated with peritoneal metastasis of GC, a semi-synthetic human scFv library (Tomlinson I) was used to isolate novel antibody fragments recognizing MKN-45, a poorly differentiated diffuse gastric adenocarcinoma cell line. Four rounds of subtractive selection each consisting of extensive pre-absorption of phage library with NIH-3T3 murine embryonic fibroblasts and AGS (a well-differentiated intestinal gastric adenocarcinoma) cell line were carried out prior to positive selection on MKN-45 target cells. ELISA-based screening of 192 phage-displayed scFv clones indicated 21 high affinity binders with specific staining of MKN-45 compared to AGS cells. Diversity analysis of the selected phage-scFvs resulted in 5 distinct sequences with multiple frequency. Further analysis by ELISA and flow cytometry verified three clones which specifically recognized MKN-45 cells. LC-MS/MS analysis of the scFv-immunoprecipitated proteins has led to identification of c-Met, HSP90 α and HSP90 β as candidate biomarkers associated with diffuse GC. Immunohistochemistry revealed capability of purified scFvs to differentiate diffuse and intestinal gastric adenocarcinoma. Taken together, the isolated MKN-45-specific scFv fragments and their cognate antigens would be beneficial in screening and management as well as targeting and therapy of the diffuse gastric adenocarcinoma.