Mesenchymal stem cells (MSCs) are attractive candidates for novel cell-therapy applications. However, the in vitro expansion of MSCs typically depends on the presence of fetal bovine serum (FBS) and coating materials derived from animal sources, which may cause contamination in clinical applications. In this study, we investigated whether human umbilical cord extract (UCE) could serve as a serum replacement and whether collagen purified from umbilical cord (UC-collagen) could act as an extracellular matrix (ECM) for the in vitro culture of MSCs derived from human UC (UC-MSCs). A total of 5.61 ± 0.54 mg UCE and 18.41 ± 2.42 mg collagen were extracted, and 1.3 ± 0.2 × 10⁵ cells were isolated from 1 g of UC, as determined by the expression of typical MSC surface markers. Importantly, the proliferation and stemness of the UC-MSCs cultured with the UCE media were similar to those cultured under FBS conditions on UC-collagen-treated plates for eight passages. Based on these results, we suggest that UCs, which are discarded as medical waste, represent a viable alternative source of xeno-free biomaterials to replace animal-derived serum and ECM materials for the cultivation of various cell types, including UC-MSCs, adipose tissue-derived MSCs, bone marrow-derived MSCs, and fibroblasts. This innovative xeno-free MSCs culture system can overcome many of the problems associated with immunogenicity, and it will further contribute to the enhancement of treatment efficiency.