Gastric cancer (GC) is an important health burden and the prognosis of GC is poor. We aimed to explore new diagnostic and prognostic indicators as well as potential therapeutic targets for GC in the current study. We screened the overlapped differentially expressed genes (DEGs) from GSE54129 and TCGA STAD datasets. Protein-protein interaction network analysis recognized the hub genes among the DEGs. The roles of these genes in diagnosis, prognosis, and their relationship with immune infiltrates and drug sensitivity of GC were analyzed using R studio. Finally, the clinically significant hub genes were verified using single-cell RNA sequencing (scRNA-seq) data. A total of 222 overlapping genes were screened, which were enriched in extracellular matrix-related pathways. Further, 17 hub genes were identified, and our findings demonstrated that BGN, COMP, COL5A2, and SPARC might be important diagnostic and prognostic indicators of GC, which were also correlated with immune cell infiltration, tumor mutation burden (TMB), microsatellite instability (MSI), and sensitivity of therapeutic drugs. The scRNA-seq results further confirmed that all four hub genes were highly expressed in GC. Based on transcriptomics and single-cell sequencing, we identified four diagnostic and prognostic biomarkers of GC, including BGN, COMP, COL5A2, and SPARC, which can help predict drug sensitivity for GC as well.