Establishing muscle lineage cell lines from fish will provide a great opportunity to study muscle development, which can eventually contribute to the improvement of the fish quality in the aquaculture industry. However, there has been a lack of the development of proper fish muscle lineage cell lines so far. Here, we report the establishment of a skeletal muscle-derived myogenic cell line from black sea bream (Acanthopagrus schlegelii). For this, we first attempted to find the optimal conditions for the primary explant culture of A. schlegelii muscle tissues and then established muscle-derived cell lines. After that, cell lines were characterized for their muscle-specific gene expression, growth, and myogenic differentiation. We found that the primary explant culture was effective when the tissue fragments were cultured in Dulbecco’s Modified Eagle Medium supplemented with fetal bovine serum and antibiotics on gelatin-coated dishes. Additionally, we confirmed that the addition of basic fibroblast growth factor was necessary to establish the cell lines. One of three cell lines established was capable of long-term culture, expressed three major myogenic regulatory genes including Pax7, MyoD, and Myog, and differentiated to myotubes in the condition using low concentration of horse serum, demonstrating that this cell line was a skeletal muscle-derived myogenic cell line.