Effect of long-term fertilization on topsoil organic nitrogen fractionation and organic carbon was explored through a 13 year-old experiment on Fluvo-aquic soil under a maize-wheat rotation system in Fengqiu County, Henan Province, China. Compared with CK (no fertilizer application) or application of mineral fertilizer, application of manure or manure mixed with mineral fertilizer significantly increased total nitrogen, acidolysable organic nitrogen, ammonium nitrogen, amino acid nitrogen, non-hydrolysable nitrogen and organic carbon. Organic nitrogen is mainly made up of ammonium nitrogen and amino acid nitrogen, and secondarily of unidentified acidolysable nitrogen, non-acidolysable nitrogen, and amino sugar, which accounts for the smallest proportion. Application of manure is especially favorable to the formation of amino acid nitrogen and non-hydrolysable nitrogen. Compared with that in the control, the proportion of acidolysable organic nitrogen to total nitrogen decreased mainly due to decreasing proportion of amino acid nitrogen and ammonium nitrogen to total nitrogen. Applying manure resulted in greater contents of total nitrogen and organic carbon than the initial contents when the experiment started. When the treatments were kept the same in NPK application rate, the application of manure mixed with mineral fertilizer increased soil N supply capacity, content of organic matter and crop yield, suggesting that it is the best fertilization method that favors environment protection and maintenance of soil fertility.