Dialdehyde celluloses (DACs) with smaller nanocrystals were prepared during the oxidization of nanocrystalline cellulose (NCC) using increased NaIO4. When NCC from the hydrolysis of microcrystalline cellulose (64 wt% H2SO4, 45 °C, 1 h) was periodate oxidized at NaIO4/anhydroglucose unit mole ratio of 5 and pH 6.5 for 5 h, the product of DAC5 can easily dissolve in water at ambient temperature. It was adopted as a bio-origin cross-linker of chitosan (CTS). The CTS films cross-linked with 1 ~ 7% DAC5 (relative to the weight of CTS in the dried film) were fabricated by the solution casting technique. FT-IR, XRD and SEM characterizations demonstrated the cross-linking reaction of DAC5 with CTS and the good compatibility between them. The dissolving and mechanical testing results showed that the cross-linked film had improved acid resistance and tensile strength compared to the pure CTS film. They could be applied in the areas of food packaging and biomedical treatments.