The invention provides a method for establishing a stable transgenic flounder embryo cell strain. The method for establishing stable transgenic flounder embryo cell strain comprises the following steps: transfecting flounder embryo cells by using plasmids which can widely express enhanced green fluorescent protein promoted by a beta-actin promoter in flounders and can express G418 resistant protein; after 24h, observing expression of the EGFP (express enhanced green fluorescent protein) under a fluorescent microscope, and through G418 screening, obtaining the embryo cells with stable genetic expression. By the method, an exogenous gene widely expressed in the flounders and promoted by the beta-actin can be effectively transfected into the flounder embryo cells, and can be genetically expressed in the flounder embryo cells stably, so that a novel method is provided for researching for flounder genes, the problem that difficult cellular-level gene function analysis and research caused by low transient transfection efficiency of the flounder cells is solved, and a novel technical means is provided for a molecular-level genetic breeding work of the flounders, and a transgenic method for stably inheriting a marine fish cell line is provided.