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Abstract Phosphorus (P) overloading is a major cause of surface water eutrophication and bottom water hypoxia. The incomplete understanding of different P pools and their corresponding bioavailability in the continuum from sources and sinks has limited the development of appropriate nutrient management strategies. Here we apply multistable isotope proxies to track colloids and identify whether specific P pools in colloids are biologically cycled at the Deer Creek‐Susquehanna River mouth stretch. Results showed that NaOH‐P_iis the most dominant P pool in the summer and winter seasons. Oxygen isotope values (δ¹⁸O_P) of NaOH‐P_iand HNO₃‐P_ipools of different size fractions of colloids are much heavier than the ranges of equilibrium values in the ambient water, which suggest that these two pools are recalcitrant against biological uptake. It further means isotopic signatures of these P pools could be used to identify the sources of colloids. Carbon (C) and nitrogen (N) isotope compositions of colloids showed that the contribution of terrestrial sources gradually decreases downstream of the river toward the bay and Deer Creek contributes disproportionately high amounts of colloids to the Susquehanna River. These findings provide valuable information on the loading of colloids and relative bioavailability of colloidal P pools in estuarine ecosystems.