As an alternative and complementary in vitro models, “cell line” provides a new platform for life science research. To a certain extent, it breaks through the limitations of ethics, law, animal welfare and animal protection in scientific research and reveals the biological effects and regulatory mechanisms of complex living organisms from a cellular and molecular perspective. Especially for endangered animals, the establishment of cell lines combined with cryopreservation technology can not only preserve the genetic germplasm of endangered animals with biological expression activity, but also provide a new platform for in vitro conservation research (such as animal toxicology experiments), which is of great significance to animal protection. Cetacean cell culture has so far proven to be a multifunctional tool for cytogenetics, virology and toxicology studies. However, from the perspective of species, tissue sources and cell types, long-term stable continuous cell lines are still relatively few. How to optimize cell culture conditions and use the cell lines to reveal more information about cetacean life mechanism is still a challenge. This paper reviews the development of in vitro models and its application in the study of cetaceans, with a view to promoting the innovation of this technology in the study of cetacean conservation.