Objective To understand the distribution pattern of the neurotransmitter 5-hydroxytryptamine (5-HT) in Clonorchis sinensis. Methods The metacercariae of C. sinensis were isolated from infected Pseudorasbora parva fish by digestion, and used to infect Kunming mice (40-50 metacercariae/mouse) by gavage. Mice were euthanized 10, 20 and 30 days after infection, and the parasites were collected from the liver portal vein. The development status of worm organs at different stages were observed after staining with acetic carmine under optical microscope, at the same time, 5-HT immunofluorescence staining was performed to observe the distribution of 5-HT using laser scanning confocal fuorescence microscope. Results Under the optical microscope, the acetic acid magenta staining showed that in the C. sinensis d₁₀ larva, the digestive and excretory organs were basically mature, the reproductive organs were not fully mature, and scattered eggs could be seen in the uterus. In the d₂₀ larva, the reproductive organs in the worm were matured, the number of eggs in the uterus increased, and the testis branches increased. In the d₃₀ adult worm, the digestive, excretory and reproductive systems were all mature, the eggs were more closely arranged in the uterus than the larvae, and the testis were enlarged and branched obviously. Under laser scanning confocal microscopy, the 5-HT immunofluorescence staining showed that in the d₁₀ larva, the fluorescence staining was strong in the central parts of the body through the system segment, nerve union and oral sucker and was distributed in a dotted pattern within the internal organs in the body. In the d₂₀ larva, a small number of nerve cells were observed in the nerve union near the ventricle sucker. Besides the central nervous system, 5-HT fluorescence staining appeared in the excretory sac, excretory pore and reproductive organs of the body. In the d₃₀ adult worm, 5-HT was found in the digestive, excretory and reproductive organs of the worm, and the fluorescence staining of the testis was stronger than that of intestinal branches and oral sucker, and there were more nerve cells in the worm. Conclusion 5-HT is widely distributed in the body of C. sinensis, mainly in the organs and viscera rich in muscle tissue. There were differences in the distribution of 5-HT in different organs at the same developmental stage, and differences were also seen in the same type of organs at different developmental stages.