The diel vertical migration of mesopelagic fishes in the southern (S-May station and S-Nov station) and central (C-Jun station and C-Dec station) South China Sea was investigated through a series of continuous field surveys conducted in May, June, November, and December 2017. These surveys employed a combination of mid-water trawl and acoustics techniques. The diel migration process, vertical distribution, acoustic migration proportion, and migration pattern of mesopelagic fishes were analyzed. The results revealed that mesopelagic fishes initiated an upward migration before sunset, with the process concluding within 30–120 min after dark. Subsequently, they commenced a downward migration before dawn, which terminated within 10–50 min after sunrise. The mesopelagic deep-sea layers of mesopelagic fishes at the S-May, S-Nov, C-Jun, and C-Dec stations ranged from 360 to 700 m, 350 to 680 m, 350 to 520 m, and 300 to 700 m, respectively. The acoustic migration proportions of mesopelagic fishes at the corresponding stations were found to be approximately 44.5%, 25.7%, 29.8%, and 58.0%, respectively. There were seasonal and regional differences in the vertical migration and distribution patterns of mesopelagic fishes in the South China Sea. A total of 228 species were identified, including 203 fish species, 23 cephalopod species, and 2 shark species. Among these, a subset of 43 fish and cephalopod species exhibited extensive diel vertical migrating behavior. Specifically, this subset comprised 23 lanternfish species, 8 cephalopod species, and 12 other fish species. Lanternfishes were the predominant diel vertical migratory species, while cephalopods also played a significant role in diel vertical migration. The diel migration behavior was found to be slight for Diaphus lucidus, Melamphaes microps, Argyropelecus affinis, and six other fish species. Non-migratory behavior was observed in Sternoptyx obscura, Argyropelecus sladeni, Sternoptyx diaphana, and 13 other fish species. The diel migration habits of 178 additional species of fish, cephalopods, and sharks could not be definitively determined.