Background: Mesenchymal stem cells (MSCs) are multipotent cells which can be isolated from many sources including umbilical cord. Isolation protocols are depended on either explant or enzymatic methods. Although fetal bovine serum (FBS) is used as a supplement in isolation and expansion of MSCs, human blood derivatives such as cord blood serum (CBS) and platelet lysate (PL) are attractive substitutes of FBS which overcome impediments of using FBS in clinical setups. Methods: Here we compared the effect of using CBS, PL and FBS supplemented media in isolation of umbilical cord tissue derived MSCs by using explant method. To do that we cultured umbilical cord tissue explant in either CBS or PL or FBS supplemented media. Isolated cells were quantified, their morphology was assessed and Cells in passage 3 were characterized based on their immunophenotyping and their potential for differentiation into adipocytes and osteocytes. Moreover, proliferation of cells was assessed by crystal violet staining. Results: All the three media succeeded to isolate MSCs and maintain their stemness characteristics. However, the highest number of isolated cells were obtained using CBS, ~10-fold more than FBS, while the number of isolated cells obtained using PL was ~2-fold more than FBS. Moreover, crystal violet showed that both PL and CBS promote proliferation of MSCs more than FBS. Conclusions: Our data suggest that, although all supplements maintain stemness characteristics of MSCs when used to isolate those cells by explant method, using human blood derived supplements is more effective than FBS. In the same context CBS is more effective than PL.