We describe a novel method for determining the degree of deacetylation (DD) of chitosans from fiber-optic measurements of refractive index. Theory analyses and the experimental setup of the method are introduced and discussed. The analytical performance of the method is described for the determination of alkaline titration end-points. Experimental results reveal that the equivalence point can be directly identified from the titration curve. The proposed method is simple, low-cost, accurate, reliable, and easy to operate for industrial application. The DD values of four chitosan samples obtained with this new method show good agreement with those yielded from 1 H NMR. Such a mechanism of refractive-index monitor should open up a new application in the field of chitosanolytic enzymes, such as chitosanase, pectinase that are important in bioprocesses.