Abstract: Portunus trituberculatus is one of the most important marine economic crab species in China, supporting coastal fisheries and regional economies; however, wild populations have experienced persistent declines in recent decades due to overfishing, habitat degradation, and environmental change, highlighting the urgent need for efficient and reliable approaches to assess its spatial and seasonal distribution. Conventional fishery-dependent surveys, such as bottom trawl investigations, are often constrained by sampling selectivity, labor intensity, and spatial coverage, which may limit their ability to resolve fine-scale population dynamics; in this context, environmental DNA (eDNA) has emerged as a promising, non-invasive tool, although its applicability to benthic crustaceans in open marine environments remains insufficiently evaluated. Here, an eDNA-based quantitative detection framework for P. trituberculatus was established and applied to the north-central coastal waters of Zhejiang Province during the summer and autumn of 2024. Species-specific primers and a TaqMan probe targeting P. trituberculatus were designed and validated, and a quantitative real-time PCR (qPCR) assay was developed for sensitive and specific detection in environmental samples. A total of 157 seawater samples were collected from 29 stations across two seasons, covering surface, middle, and bottom water layers, and bottom trawl surveys were conducted concurrently to provide fishery-independent reference data. The developed primers and probe exhibited high specificity, with no cross-amplification detected in closely related crustacean species. Horizontally, P. trituberculatus eDNA displayed pronounced spatial heterogeneity in both seasons, with consistently elevated concentrations in the Yushan Fishing Ground, indicating this area as a key habitat and aggregation zone. Vertically, distribution patterns differed between seasons: in summer, eDNA concentrations did not differ significantly among layers, suggesting strong vertical mixing and rapid dispersion, whereas in autumn, eDNA concentrations were significantly enriched in the middle layer and clear stratification was observed, reflecting seasonal changes in water-column stability and species behavior. Furthermore, both the mean eDNA concentration and detection rate in autumn were significantly higher than those in summer, consistent with seasonal migration and increased population aggregation. Compared with bottom trawl surveys, eDNA analysis provided a more comprehensive indication of species presence and distribution, particularly where trawl efficiency may be limited; the combined application of eDNA monitoring and trawl surveys enabled a refined assessment of the spatial and seasonal distribution patterns of P. trituberculatus. Overall, this study demonstrates the effectiveness and sensitivity of eDNA techniques for monitoring benthic crab resources in open marine systems and provides a robust scientific basis for resource assessment, ecological conservation, and the sustainable management of P. trituberculatus fisheries.