Hydrolysis of cellulose in concentrated phosphoric acid is known to give two distinctive mono-disperse cello-oligomers: the degree of polymerization (DP) of 7 and 15. To understand the formation mechanism of monodisperse cello-oligomers, the transition of DP during phosphoric hydrolysis was monitored by size-exclusion chromatography combined with multiangle laser-light scattering analyses (SEC/MALLS). The obtained results suggested that not the hydrolysis at a specific length but random hydrolysis was likely. The rate of hydrolysis slowed down when the DP reached around 40, and the pool of cello-oligomers appeared at the incubation of 35 days. Then, the mono-disperse cello-oligomers were recovered from the pool of cello-oligomers by fractionation based on the solubility difference.