Cellulose nanocrystal (CNC)-reinforced bio-composite films containing glycerol were produced using the solution casting technique. The influences of the addition of CNC (2, 4, and 8 wt%) and glycerol (10, 20, and 30 wt%) on the properties of the bio-composite films were studied in the present work. The resulting films were characterized by X-ray diffraction (XRD), Fourier transform infrared (FT-IR) spectroscopy, and thermogravimetry analysis (TGA), and according to their tensile, water absorption, and light transmission behavior. The introduction of 4 wt% CNC into the chitosan film did not affect the thermal stability, but the presence of 20 wt% glycerol reduced the thermal stability. The addition of 4 wt% CNC to the chitosan film increased its tensile strength, tensile modulus, and elongation at break by 206%, 138%, and 277%, respectively. However, adding more than 8 wt% CNC resulted in a drastic reduction in the strength and ductility of the chitosan film. The highest strength and stiffness of the chitosan bio-composite film were attained with 4 wt% CNC and 20 wt% glycerol. The water absorption and light transmission of the chitosan film were reduced dramatically by the presence of both CNC and glycerol.