<p>Three-dimensional (3D) cell culture models represent an important advancement in fish studies, offering significant improvements over traditional two-dimensional (2D) methods. While 2D cultures remain common in aquaculture research, compelling evidence suggests that 3D techniques provide more physiologically relevant microenvironments by better replicating in vivo tissue architecture and cell-cell interactions. When performing 3D cell culture experiments with fish cells, the cellular environment can be manipulated to closely mimic natural conditions, providing more accurate data about intercellular communications, disease mechanisms, drug responses, and metabolic processes. Various techniques are employed, including scaffold-based systems (hydrogels, polymeric materials), scaffold-free methods (spheroids, bioprinting), and complex structures (organoids, bioengineered organs), each offering distinct advantages for specific applications. While 3D culture systems have been extensively advanced for mammalian cell research, their use in fish cell studies relevant to aquaculture is still limited, with existing efforts largely focused on model organisms like zebrafish and rainbow trout. 3D cell culture has the potential to bridge the gap between traditional cell culture and live animal models in aquaculture research, enabling more relevant physiological studies and reducing the need for animal testing. The present review compares 2D and 3D fish cell culture approaches, details various 3D culture techniques applicable to aquatic species, and examines both current applications and future potential of these advanced culture systems in expanding aquaculture research.</p>