The design of duplicated samples constitutes the primary stage in the standardisation of eDNA monitoring processes. A plethora of studies have hitherto investigated the optimal number of duplicated samples required for the purpose of ascertaining the precise eDNA information at a given sampling site or transection. However, the question of whether duplicated samples should be collected at a series of sites or in continuous moments remains unresolved in the context of eDNA monitoring at this scale. This issue is of particular significance for the practice of eDNA monitoring. To optimize the sampling strategy of duplicated samples in eDNA monitoring of large rivers, the present study employed a single-variable controlled experiment in the Wuhan section of the Yangtze River. A total of 16 eDNA samples were obtained from 27 June to 14 July 2022 on a daily basis (temporal group samples) and 16 eDNA samples were collected across the Yangtze River on 28 June and 12 July 2022 (spatial group samples). The composition of the detected species and OTUs in each eDNA sample was then analyzed. The following steps were taken in order to provide suitable suggestions for how to set duplicated samples in a small spatiotemporal scale eDNA monitoring practice in a large river. Firstly, the species and OTU compositions, temporal heterogeneity of temporal group samples, and the species and OTU compositions, spatial heterogeneity of spatial group samples, were quantified. The findings demonstrated that, for bacteria and metazoa, the total number of species detected in spatial group eDNA samples exceeded that detected in temporal group eDNA samples. Furthermore, the spatial heterogeneity of species detected in eDNA monitoring was found to be greater than the temporal heterogeneity of the same. In contrast, an antithetical status was observed for the three taxonomies of fungi, algae and protozoa. In essence, the study proposed that in the context of monitoring environmental bacteria and aquatic metazoa within a specified sampling site or transection of a substantial river, the implementation of spatial duplicated sampling of eDNA monitoring should be accorded priority in the design of duplicated samples. With regard to the monitoring of fungi, algae and protozoa, the implementation of temporal duplicated sampling of eDNA monitoring should be prioritised within the framework of duplicated samples design. It is evident that the conclusions drawn must be subject to verification, given the potential variations in the community structure of aquatic organisms across different rivers, sections and temporal periods. It is imperative to emphasise that particular attention must be allocated to the selection of sampling time when implementing spatial duplicated sampling, and the selection of sampling point when executing temporal duplicated sampling. Furthermore, it may be necessary to consider the replication of samples when the focus is on the monitoring of a subdivision taxonomy.