A gas chromatography-mass spectrometry (GC–MS) method for the determination of difenidol hydrochloride in biological specimens has been developed. The method exhibited excellent recovery (> 90%) and precision (RSD < 10%), and the LOD was 0.05 μg/mL or μg/g, which met the requirements of bioanalytical method. Through the animal model of the forensic toxicokinetics, the dynamic distribution, postmortem redistribution (PMR) and stability in specimen preservation process of difenidol in animals were studied. The experimental results showed that after intragastric administration, the difenidol’s concentrations in the heart-blood and various organs increased over time except stomach, and then decreased gradually after reaching the peaks of concentration. The toxicological kinetics equation and toxicokinetic parameters were established by processing the data of the mean drug concentration of difenidol changing with time. In PMR experiment, the concentrations of difenidol in some organs closer to the gastrointestinal tract (heart-blood, heart, liver, lung, kidney, and spleen) changed significantly at different time points. But the concentration of difenidol in brain tissues which were far away from the gastrointestinal tract and muscles with larger overall mass was relatively stable. PMR of difenidol was therefore confirmed. Thus, the effect of PMR on the concentration of difenidol in the specimens should be considered in cases involving difenidol poisoning or death. Furthermore, the stability of difenidol in heart-blood samples from poisoned rats was investigated at various time points and under different preservation conditions (20 °C, 4 °C, − 20 °C and 20 °C (1% NaF)) for a period of two months. Difenidol was stable and did not decompose in the preserved blood. Therefore, this study provided the experimental basis for the forensic identification of the cases of difenidol hydrochloride poisoning (death). PMR has been verified by practical lethal cases.