[Abstract] Objective: To compare the culture characteristics of human umbilical cord mesenchymal stem cells (hUC-MSCs) cultured in two different surface-material flasks. Methods: Ten fresh umbilical cords were collected, and umbilical cord tissue was isolated using the tissue block method and cultured adherently for 12 days. Primary cells were collected, and 2×10^6 cells were respectively seeded into 225 cm2 high-adhesion flasks (Group A) and 225 cm2 ordinary flasks (Group B) for culture for 72 hours until passage 1 (P1). P1 cells were collected and further cultured for 72 hours until passage 2 (P2). The experiment was repeated five times. The morphology of cells at P1 and P2 in both groups was observed under a microscope, and P2 cells were compared for digestion duration, cell growth curves, surface marker expression, and trilineage differentiation potential. Cells were also cultured to P4 to compare the SA?β-galactosidase positivity rate. Results: Cells in both groups displayed a flat spindle-shaped morphology. The digestion time of P2 cells in Group A was (123.8 ± 3.09) seconds, while in Group B it was (38.5 ± 2.20) seconds, showing a statistically significant difference (P<0.001). The cumulative cell amplification from P0 to P2 in Group A was (129.49 ± 0.89)-fold, significantly higher than that in Group B [(101.4 ± 1.67)-fold, P<0.001]. The SA?β-galactosidase positivity rate in Group A was (2.58 ± 0.44)%, significantly lower than in Group B(4.79 ± 0.33)%, and both met the quality standards for mesenchymal stem cells. Conclusion: Under the same seeding density, high-adhesion flasks yield higher cell amplification, lower cell senescence, and optimize the culture system and efficiency, thereby improving cell quality.