The present work focused on modifying the chitosan structure which has noticeable places in advanced research. The effect of UV-ozone irradiation with different exposure doses (9.68 x 10-14, 19.35 x 10-14, 29.03 x 10-14 and 38.75 x 10-14 J/m2) on the microstructural, thermal stability and optical properties of chitosan was studied. X-ray diffraction (XRD), selected area electron diffraction (SAED), high-resolution transmission electron microscopy (HRTEM), optical parameters techniques as well as Differential thermal analysis (DTA) and thermogravimetric analysis (TGA and DTG), were performed. The obtained results indicated that, effects on semi-crystalline structures have occurred. Detected variations in XRD, SAED, HRTEM, DTA and TGA analyses under investigation may be attributed to the different degrees of crystallinity, induction of structural changes and molecular configuration of chitosan upon exposure to UV-ozone. The results confirmed that the UV-ozone irradiation plays a role in refining the microstructure and changes occurring in chitosan matrix for future biomedical applications.