Few studies have investigated the extractable organic nitrogen (EON) formation mechanisms, and the sources of EON have long been debated. Using ¹⁵N labeling, we performed a 120-day laboratory incubation experiment to explore the dynamic contributions of different types of added N (ammonium-N, ryegrass-N and their combination) to soil EON and the role that microorganisms play in N transformation into EON. We show that the ¹⁵N abundances and recoveries in soil EON pool were relatively low during the incubation, except the first hours after ryegrass addition in ¹⁵N-ryegrass addition treatments. In general, most of the EON during the incubation was soil derived, and both ammonium-N (80 mg kg^-1 ) and ryegrass-N (160 mg kg^-1 ) additions made minor contributions (3–4% and 8–13% during day 1–120) to the soil EON pool. Moreover, along with the decline in ¹⁵N recoveries in microbial biomass nitrogen (MBN) pool, the lost MB¹⁵N did not enter into the EO¹⁵N pool. Our study demonstrates 1) that EON is a stable N pool in agricultural soil and is less affected by exogenous N addition and 2) that microbial N uptake and release processes contribute little to the soil EON pool.