Abstract: Normalized and systemic eDNA (environmental DNA) monitoring is very helpful for aquatic biodiversity monitoring, assessment and conservation. The lack of quantified spatial resolution of eDNA monitoring hinders the implement of normalized and systemic eDNA monitoring. To quantify the spatial resolution of eDNA monitoring, we explored a quantification method based on black-box model, simplified ecological processes and the statistical representation of spatial resolution. e took a case study of quantifying the spatial resolution of eDNA monitoring in the middle reach of Yangtze River. Th30 sampling transections with an approximate 30 km  ccording to eDNA sampling in mean-flow period, next generation sequencing and watershed biological information flow (WBIF) analyzing, the characteristics of eDNA monitoring spatial resolution. Here, two taxa (eukaryotes and prokaryotes, respectively indicated by mitochondrial COI gene and 16S rRNA gene) were identified and analyzed. Results showed that , the prokaryotic WBIF transport capacity was 99.91%/km, the WBIF labeling nonliving prokaryotic materials accounted for 23.83% of the total prokaryotic WBIF and had a half-life distance of 48.45 km; the eukaryotic WBIF transport capacity was 99.85%/km, the WBIF labeling nonliving eukaryotic materials accounted for 67.93% of the total eukaryotic WBIF and had a half-life distance of 30.00 km. There was tradeoff between the reliability and coverage of eDNA monitoring spatial resolution. The reliability and coverage of prokaryotic eDNA monitoring spatial resolution got their balance point at 39 km with the value approximate 86%. The reliability and coverage of eukaryotic eDNA monitoring spatial resolution got their balance point at 28 km with the value approximate 65%. To adapt different monitoring aims, one could select different eDNA monitoring spatial resolutions based on a suitable reliability or coverage. To identify the species composition of a unit reach, one should give priority to the coverage of eDNA monitoring spatial resolution. 90% prokaryotic coverage need a spatial resolution of 27 km (reliability, 84.18%); 90% eukaryotic coverage need a spatial resolution of 6 km (reliability, 41.38%); 80% eukaryotic coverage need a spatial resolution of 13 km (reliability, 50.64%). To identify the species spatial heterogeneity of a set of adjacent reaches, one should give priority to the reliability of eDNA monitoring spatial resolution. 90% prokaryotic reliability need a spatial resolution of 58 km (coverage, 82.30%); 90% eukaryotic reliability need a spatial resolution of 78 km (coverage, 38V61%); 80% eukaryotic reliability need a spatial resolution of 50 km (coverage, 49.70%). As a pilot study of eDNA monitoring spatial resolution in middle Yangtze River, thresults were not accuracy enough, because of the spatial and temporal heterogeneous WBIF along the river, and the systemic errors of random sample. Thwork could provide a quantitative reference for the eDNA monitoring sampling setting in the middle reach of Yangtze River and a methodology reference for the eDNA monitoring spatial resolution estimating in other free flowing rivers and reaches.