Abstract: Species distribution monitoring and biomass assessment are crucial for the management and conservation of fishery resources. Compared to traditional fishery resource surveys, the environmental DNA (eDNA) method is an eco-friendly biological monitoring method that can monitor species without harming organisms. In this study, specific primers and TaqMan probes were designed to analyze the horizontal (44stations) and vertical distribution (175 water samples) of Sepiella japonica in the East China Sea using eDNA. The eDNA concentrations were examined in relation to environmental variables, including total depth, sampling depth, temperature, salinity, dissolved oxygen, turbidity, chlorophyll, phosphate, silicate, nitrate, \rmNH^+_4 and \rmNO^-_2 . The results showed that the eDNA detection rate of S. japonica was 17.14%, with no significant difference among stations, but a significant difference in vertical distribution (P<0.05). High concentrations of S. japonica eDNA were detected in mid-surface water, while no eDNA was detected at depths greater than 81 m. In addition, the eDNA concentration (Ln(eDNA+1)) and presence/absence (YNeDNA) of S. japonica were affected by environmental factors such as total depth, turbidity, salinity, dissolved oxygen, silicate, \rmNH^+_4 , and Nitrate. Our study proves that eDNA is a reliable method, which can be used as a supplement to traditional resource investigation. It offers technical support for further research on the resource distribution and habitat selection of S. japonica in the East China Sea, and provides a reference for resource restoration and sustainable management efforts.