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Cellulose is an attractive biomolecule that exhibits irreversible intermolecular interactions and forms assemblies with high stability and robustness. Nevertheless, its tendency to irregularly aggregate prevents the polysaccharide chains from assembling into a fine structure. Herein, we demonstrate the formation of gels with relatively homogeneous structures via the self-assembly of cello-oligosaccharides modulated by organic solvent additives or elevated temperatures. Cello-oligosaccharides in a dissolved state in an aqueous alkaline solution started to self-assemble upon the addition of an acid for neutralization at room temperature, forming hydrogels with heterogeneity at the sub-micrometer or larger scale. On the other hand, cello-oligosaccharide assembly in the presence of 20% (v/v) water-miscible organic solvents produced approximately ten times stiffer gels with more homogeneous structures. The highest Young's modulus of the gels in this study was ~ 1.5 kPa. Moreover, increasing the assembly temperature from 25 °C to 50 °C also increased the gel stiffness. It was suggested that organic solvent additives and elevated temperatures decreased the solubility of cello-oligosaccharides and thus increased the assembly kinetics for the formation of more homogeneous network structures before non-organized aggregation. These findings promote the development of self-assembled cellulose and cello-oligosaccharide materials with organized structures. [ABSTRACT FROM AUTHOR]