Two chitooligosaccharide samples (COS1 and COS2) were prepared by enzymatic degradation of chitosan. The molecular weight of the main component was determined by mass spectrometry to be 2340 and 4303, respectively. They showed cholesteric liquid crystal structures in concentrated solutions. The critical concentrations to form a lyotropic liquid crystalline phase in formic acid were measured by means of polarized optical microscopy, refractometry and Fourier transform infrared spectroscopy. The results from the three methods agreed with each other. The experimental value of COS2 was quantitatively consistent with that calculated according to Khokhlov and Semenov theory. But the experimental value of COS1 diverged from the theoretical one, which is explained by the inaccuracy at higher concentrations of the second virial approximation underlying this theory.