Fish cell lines are essential tools in aquaculture research, offering platforms for studying viral pathogens, vaccine development, and toxicology. They aid in understanding immune responses, host–pathogen interactions, and cellular processes, contributing to disease management and sustainable aquaculture. Fish cell cultures have emerged as an effective biological alternative to using animals in research. An extensive number of 960 fish cell lines have been documented by Cellosaurus, an encyclopaedia of cell lines as of January 2025. These in vitro models facilitate vaccine development, drug screening, and pollutant assessment, reducing reliance on live animals while ensuring reproducible results. Beyond experimental trials, fish cell cultures are cost-effective alternatives to mammalian systems for recombinant protein production, and vaccines. Techniques like CRISPR/Cas9 and 3D cultures enhance their therapeutic potential. The use of unauthenticated cell lines would result in the generation of spurious data, and the International Cell Line Authentication Committee (ICLAC) - Register of Misidentified Cell Lines currently lists 593 cell lines. However, challenges such as limited species-specific cell lines, authentication issues, and cross-contamination persist, emphasizing the need for standardized protocols and expanded repositories to maximize their research and industry applications. Advancing fish cell line research and addressing existing challenges will unlock their full potential, driving innovations in aquaculture and biotechnology.