Soil organic nitrogen (SON) and its mineralization are among the most critical factors that affect soil N availability. However, it is unclear how crop residue return and chemical N fertilization affect soil N availability by altering SON and its mineralization. Therefore, we studied the SON pools, SON mineralization, and related soil properties and enzymes activities in the lower reaches of the Yangtze River Valley in China with stand ages of 7‐year N (0, 75, 150, and 300 kg N ha−1) fertilization and 4‐year barley‐cotton residue return. Regardless of residue retention or removal, the contents of total SON and its fractions (ammonium N, amino acid N, amino sugar N, and unidentified N) increased consistently with the increasing of N input, while SON mineralization, microbial biomass N content, urease and protease activities showed a curvilinear response that was highest at N rate of 150 kg N ha−1. Crop residue returning significantly increased SON content under 75 and 150 kg N ha−1, mostly due to the increase of amino acids N. Other variables, such as SON mineralization, microorganism, and enzymes, were also enhanced by residue returning, and the largest increases were observed at 150 kg N ha−1. In addition, our result also show that excessive N input (300 kg N ha−1) would reduce soil N availability by mainly suppressing microbial N processes. We concluded that crop residue combined with appropriate N has a great potential to improve soil N availability by mainly improving amino acid N content and SON mineralization.