Sea buckthorn (Hippophae neurocarpa), a plant with both medicinal and edible properties, exhibits high content of serotonin with a unique brown color. Here, we assemble the 1002.54 Mb genome sequence of H. neurocarpa and find that it has evolved from two sequential polyploidizations with four sub-genomes. Based on in vitro enzyme activity and transient overexpression experiments, we confirm that the expression of both HnT5H1 and HnT5H2 genes from tandem duplication contributes to high accumulation of serotonin in sea buckthorn. Furthermore, we verify that the HnGLK1 gene plays a crucial role in continuous chlorophyll accumulation, driving the brown color formation of H. neurocarpa fruit. Collectively, the high-quality genome of H. neurocarpa offers valuable insights into the mechanisms underlying serotonin biosynthesis and abnormal coloration and serves as a valuable resource for further functional genomic studies and molecular breeding efforts in sea buckthorn.