In this study, concentrated polymer brush-modified cellulose nanofibers (CNFs) with different fiber lengths were used for the flocculation of cells for systematically studying the mechanism of this unique cellular flocculation based on colloidal flocculation theory. Concentrated poly(p-styrenesulfonic acid sodium salt) brush-grafted CNF (CNF-PSSNa) with different fiber lengths were cultured with three different cell types to examine their influence on floc (cell clusters formed by cellular flocculation) characteristics. The floc size and survival rate could be controlled by modifying the CNF-PSSNa fiber lengths. The three cell types showed the same flocculation tendency after culture, indicating the applicability of the method in different cell lines. After 2 weeks of culture, CNF-PSSNa increased the specific expression of hepatocytes compared to the two-dimensional cell culture. Thus, owing to its wide applicability, high cell viability, and ability to control cell size and improve cell function, this technology could be used as a new three-dimensional cell culture method.