Abstract <p>The golden yellow phenotype of the large yellow croaker <i>Larimichthys crocea</i> is due to the distribution of light-absorbing xanthophores and melanophores. The scales of the croaker are covered with a pigment cell layer. Here, we present a protocol for isolating pigment cells from pigment cell layer attached to the scales adopted for the large yellow croaker. At first step, scales with chromatophores attached were treated with trypsin solution digestion for 20 min and collagenase I solution digestion for 2 h. After obtaining single-cell suspensions, individual xanthophore and melanophore were collected using a microinjection needle equipped with Cell Tram oil pressure microinjection apparatus. The transcriptomic analysis of xanthophores and melanophores samples showed distinguishable expression pattern of gene markers of pigment cells. Additionally, <i>in vitro</i> treatment with α-melanocyte-stimulating hormone induced xanthosomes dispersion in isolated xanthophore. Thus, we present an optimized protocol for isolating pigment cells from the skin of large yellow croaker, enabling a single cell approach in future studies focused on this area.</p>