Gemcitabine liposome injection (stealth liposomes) has facilitated the targeting of gemcitabine for cancer treatment. We systemically review liposome-based drug-delivery systems, which can improve pharmacokinetics, reduce side effects and potentially increase tumor uptake, for pancreatic cancer therapy. A novel liposomal formulation, which allows for higher tumor targeting efficiencies and can be used in current clinical trials to treat this challenging disease, has gained great popularity and attention. In this study, since extrusion technology was used to make sterile preparation of liposomes, the process included aseptic production process and sterile filtration. During the preparation, it has been found that the lipid concentration, emulsification speed and time, the homogenization times and pattern, the lipid solution temperature are all critical parameters for the character of the gemcitabine liposome injection. The particle size method and zeta potential method to characterize a PEGylated liposomal drug formulation of the anti-cancer agent gemcitabine was developed. The methods are specific, precise, reproducible and sensitive, therefore they are suitable for the determination of particle size and zeta potential of gemcitabine liposome injection. Negative staining technology of transmission electron microscopy revealed that gemcitabine liposome injection has a typical morphology, which enables liposomal surfaces could be seen so additional visual information on the stealth liposome can be routinely obtained in a fast and reliable manner. Moreover, the above three methods are simple, fast and would be used for continuous quality control of gemcitabine liposome injection when it moves to cGMP production scale.