Abstract: As a major trans-basin water transfer project, the water quality safety of the Middle Route Main Canal of the South-to-North Water Diversion Project is of paramount importance. This study collected surface sediment and water samples from 14 siltation zones along the canal, systematically analyzed physicochemical parameters of the water and nutrient content in sediments, characterized six phosphorus forms, evaluated the release potential of endophytic phosphorus, and revealed the process of phosphorus deposition in water bodies. The results demonstrated that algae absorb phosphorus from the water and deposit it into sediments upon death, interacting with geochemical processes to drive phosphorus accumulation in sediments. This results in a phosphorus-limited state in the canal water (average N/P molar ratio of 1203.2), with total phosphorus in sediments predominantly consisting of organic phosphorus and iron-bound phosphorus, and bioavailable phosphorus accounting for 65.8%. Spatially, phosphorus levels show a trend of higher concentrations in the middle and upper reaches and lower concentrations in the downstream. Single-factor pollution indices indicate that most monitoring points are at clean or normal levels, while the bioavailable phosphorus index reveals moderate to severe enrichment at multiple points in the middle and upper reaches, indicating high release potential of phosphorus in sediments. It is recommended to implement water quality regulation through two approaches: interface redox regulation and calcium-phosphorus transformation, to effectively ensure the water quality safety of the Middle Route Main Canal of the South-to-North Water Diversion Project.