By establishing a viable and efficient technique for culturing hypothalamic neurons in vitro, in order to lay the foundation for studying the feeding mechanism and energy metabolism of mandarin fish, the hypothalamus of mandarin fish was isolated and digested into single cell suspension by type I collagenase. The cell morphology was observed on the 3rd, 4th, 5th and 6th day of culture and the results showed that on the third day of culture, the number of cells was small, the cell body was small and single distribution; on the 4th day of culture, the number of cells increased significantly, with typical neuron morphology and full cell body; on the 5th day, the fusion degree of the cell body increased, the bulge increased, and many branches connected with each other to form a dense network of nerve fibers; on the 6th day, the activity of cells decreased and apoptosis began. CCK-8 method was used to detect the cell activity. The results showed that the cell activity was the highest on the 5th day of culture. Two methods were used to identify neurons, the RT - PCR showed that cultured hypothalamic cells could express noggin, and the purity of neurons in hypothalamus was 95.9% by immunofluorescence. These results show that the neurons in the hypothalamus of mandarin fish with high purity can be obtained by this culture method, which lays a foundation for further study on the feeding mechanism and energy metabolism mechanism of mandarin fish at the cell level.