Gastric cancer is one of the deadliest malignant tumors of the digestive tract, and its development and metastasis are regulated by various factors. Some studies have shown that PSMD2 is involved in cancer development by regulating the tumor microenvironment stiffness. However, the exact mechanism is unclear, and effective means to quantify the effect of PSMD2 on gastric cancer tissue hardness are lacking. Herein, we revealed the mechanical heterogeneity of tumor tissues in gastric cancer patients using a large-scale AFM-based in situ method. Gastric cancer cryosections were probed by this method under aqueous condition. The in situ fluorescence images were measured to correlate tissue stiffness with PSMD2 expression. Experimental results clearly revealed the specific distribution of mechanics in gastric cancer tissues under differences in PSMD2 expression. The study unveils the effect of PSMD2 expression levels on cancer invasion and increased matrix stiffness, providing a novel insight into gastric cancer research.