The oriental fruit fly (Bactrocera dorsalis) is a serious agricultural pest, particularly during its larval stage, when it poses significant threats to crops and leads to reduced agricultural yields. Serotonin (5-HT), an important neurotransmitter, regulates various behaviors in insects, including feeding. Therefore, investigating the serotonergic neural network in the brains of B. dorsalis larvae is crucial for clarifying the specific mechanisms underlying behavioral regulation and for developing behavioral modulators targeting this pathway. In this study, we performed immunohistochemical staining of the nervous system of third-instar B. dorsalis larvae using synapsin and serotonin antibodies, followed by analysis of the structural features and distribution patterns of serotonergic neurons using laser confocal microscopy. Our results indicated that the central nervous system of B. dorsalis larvae primarily consists of the brain and thoraco-abdominal ganglia, which were interconnected and exhibit a bilateral symmetrical arrangement. We found that the neuropils in the brain were underdeveloped, with the gnathal ganglion and thoracic ganglia each containing 3 neuromeres, while the abdominal ganglion comprising 8, with distincted boundaries between each neuromere. A total of 86 serotonergic neurons were identified in the brain of third-instar larvae, including 20 in the central brain, 22 in the gnathal ganglion, and 44 in the thoraco-abdominal ganglia. The 20 cell bodies in the central brain could be further classified into four clusters, each containing 1 to 3 cell bodies. Additionally, 6 commissures connecting the two brain hemispheres were observed. The SE2 and SE3 cell body clustered in the gnathal ganglion project neurites to form commissures. The serotonergic neurons were symmetrically arranged on both sides of the thoraco-abdominal ganglia, extending neurites outward to form additional commissures. This research revealed the distribution characteristics of serotonergic neurons in the nervous system of B. dorsalis larvae, providing an anatomical foundation for understanding how this biogenic amine regulates behavior. It also offers insights into the development and assembly of serotonergic neurons in dipteran insects.