Recent progress has led to the identification of liver stem/progenitor cells as suitable sources for generating transplantable liver cells. However, the great variability in methods utilized to isolate liver stem/progenitor cells is a considerable challenge for clinical applications. The polyelectrolyte-multilayer technique can constitute a useful method for selective cell adhesion. Whether enrichment of liver stem/progenitor cells can be achieved utilizing polypeptide polyelectrolyte-multilayer films was investigated in current work. Fetal liver cells isolated from E13.5 mouse embryos were seeded on the poly-l-glutamic acid/poly-l-lysine alternating films, and we revealed that fetal liver stem/progenitor cells were selected and formed colonies. These undifferentiated colonies were maintained on the films composed of four alternating layers, with the topmost poly-l-glutamic acid layer judged by the constitutive expression of stem-cell markers such as Dlk-1, CD49f, and CD133 and self-renew marker-beta-catenin. Our work has demonstrated that highly tunable polyelectrolyte-multilayer films were suitable for selective enrichment of liver stem/progenitor cells in vitro.