Cell-cultured meat production relies on stable, proliferative seed cells, commonly sourced from muscle satellite cells (MuSCs) and adipose-derived mesenchymal stem cells (AD-MSCs). However, establishing such cell lines in fish species remains technically challenging. While pluripotent stem cells (e.g., ESCs/MSCs) offer alternatives, their differentiation efficiency and predictability are limited. Here, we developed TCCF2022, a novel caudal fin-derived cell line from Topmouth culter (Culter alburnus), which expresses pluripotency markers (AP, Oct4, Sox2, Klf4, and Nanog) and aggregated growth to form 3D spheroids. Forskolin supplementation enhanced pluripotency maintenance. The presence of adipogenic and myogenic lineage cells within the 3D spheroids was confirmed, demonstrating their potential as seed cells for cell-cultured meat. Using a small-molecule cocktail 5LRCF (5-Azacytidine, LY411575, RepSox, CHIR99021, and Forskolin), we successfully differentiated TCCF2022 cells into functional myotubes. Additionally, we established a method to induce the differentiation of TCCF2022 cells into adipocytes simultaneously. Thus, the TCCF2022 cell line can be used to improve muscle fiber formation and lipid composition, potentially enhancing the nutritional profile and flavor of cultured fish meat.