This study aimed to optimize the ultrasound-assisted enzymatic extraction process of selenium-enriched dark tea polysaccharides (STP) and investigate their antitumor mechanisms. Single-factor experiments and Box-Behnken response surface methodology were employed to systematically optimize the extraction conditions. The results showed that the optimal extraction parameters were as follows: cellulase dosage of 2.10%, solution pH of 5.00, and ultrasonic temperature of 45.0 ℃, achieving a polysaccharide yield of 11.03%. Cellular experiments demonstrated that STP significantly inhibited the proliferation and migration of hepatocellular carcinoma Huh7 cells in a concentration-dependent manner. Transcriptome sequencing analysis revealed that STP effectively suppressed the Wnt/β-catenin signaling pathway by precisely downregulating key transcription factors, lymphoid enhancer-binding factor 1 (LEF1) and FBJ murine osteosarcoma viral oncogene homolog (FOS), thereby fundamentally curbing the abnormal proliferation and invasive capabilities of tumor cells. Furthermore, STP inhibited angiogenesis and extracellular matrix remodeling by downregulating critical genes such as vascular endothelial growth factor C (VEGFC) and lipoxygenase (LOX), thereby interfering with tumor cell migration and invasion processes. These findings provide theoretical support for the application of STP in the development of bioactive polysaccharides and functional foods.