Chitosan, CS, cross-linked with bivalent palladium has shown enhanced mechanical and thermal properties depending on the transformation of the structure at a microscopic scale. In the present study, CS directly cross-linked by palladium cation membranes (CS-cr-PM) was prepared through a solution-casting method. Mobility of chitosan chains were greatly reduced after crosslinking, making a great reduction in the swelling ratio studied by a water-swelling degree measurement, which led to an improvement in molecular chain rigidity. In order to investigate the chain packing at the molecular level in the ionic cross-linked CS system, the structure of chemically-crosslinked CS is investigated by means of the combined use of wide angle X-ray diffraction (WAXD) and infrared measurements, and a combination of positron annihilation lifetime spectroscopy (PALS) and simultaneous coincidence Doppler broadening (CDB) spectroscopy offers coherent information on both the free-volume related sub-nano level molecular packing and the chemical surrounding of free volume nanoholes in CS-cr-PM as a function of palladium salt loading. The variations in the free volume size and size distribution have been determined through the ortho-positronium (o-Ps) lifetime and its lifetime distribution. The studies showed that a strong interaction between CS molecules and palladium cations results in the change in crystallinity in formed CS-cr-PM leading to variational chain packing density. Meanwhile, significant inhibition effects on positronium formation due to doping are observed, which could be interpreted in terms of the existence of chloride ions. Applications of positron annihilation spectroscopy to study the microstructure and correlated positron annihilation characteristics of an ionic cross-linked CS system are systematically discussed.