Ex vivo expansion of hematopoietic stem cells (HSCs) is very important for clinical applications of cord blood (CB). With the aim to find proper culture duration for ex vivo expansion, mononuclear cells (MNC) was applied as starting culture cells to expand HSCs and the repopulating potential of seven-day and fourteen-day cultured $CD34^+$ cells were compared. The average expansion of total cells and $CD34^+$ cells cultured for 7 days were higher than those cultured for 14 days. The results of phenotypic analysis of fresh and cultured cells showed that the percentage of $CD3^+$ cells declined and the percentage of $CD33^+$ cells increased during culture. The engraftment levels of fourteen-day cultured $CD34^+$ cells were higher than those of fresh and seven-day cultured $CD34^+$ cells. Fourteen-day cultured $CD34^+$ cells also showed better multilineage reconstitution ability than fresh and seven-day cultured $CD34^+$ cells. The results of the present study demonstrated that prolonged culture could preserve the hematopoietic reconstitution ability of ex vivo cultured CB cells and improve the engraftment level in NOD/SCID mice.