As an important grain production area in China, the Northeast Black Soil Region has experienced many problems, such as soil degradation, fertility decline, and grain yield reduction, in recent years. Optimizing fertilizer management is an important measure to maintain and enhance soil fertility. However, improper fertilizer application could aggravate nutrient losses and greenhouse gas NO emissions, thus leading to soil degradation and environmental pollution. The objectives of the present study were to investigate the response of NO emission from black soil to long-term application of organic and chemical fertilizers and the key controlling factors. Soil samples （0-20 cm） were collected from a total of nine treatments, including organic fertilizer as the primary treatment （M- no organic fertilizer； M- low organic fertilizer； M- high organic fertilizer） and chemical fertilizer as the secondary treatment （CK- no fertilizer； N- chemical nitrogen fertilizer； NPK- chemical nitrogen, phosphorus, and potassium fertilizer）, in a long-term experiment （32 years） on the black soil of Gongzhuling, Jilin Province. The soil samples were incubated at 25℃ with 65% field water holding capacity for 21 days, and NO emission and soil physico-chemical biological properties were determined. The results showed that long-term application of organic and chemical fertilizers notably increased NO emissions from black soil. Compared to those from the MCK treatment [（0.25±0.01） mg·kg, in terms of N, the same as below], the cumulative NO emissions from the only organic fertilizer treatment significantly increased by 361%-456% [（1.17±0.02） mg·kg and （1.41±0.02） mg·kg for the MCK and MCK treatments, respectively]. Furthermore, the NO emissions strongly increased with increasing organic fertilizer application amounts. Cumulative NO emissions were significantly higher in the chemical fertilizer treatments by 96%-236% [（0.49±0.01） mg·kg and （0.84±0.03） mg·kg for the MN and MNPK treatments, respectively] compared to those in the MCK treatments. Moreover, the increased NO emissions due to fertilizers application were significantly larger in the MNPK relative to MN treatments. The positive effects of chemical fertilizer application on NO emission decreased under organic fertilizer amendments （M and M）, indicating that organic fertilizer application alleviated increased NO emission because of chemical fertilization. The application of organic fertilizers significantly increased bulk soil, aggregate organic carbon （SOC）, total nitrogen （TN）, and soil microbial carbon and nitrogen contents. The application of organic combined with chemical fertilizers further increased SOC and TN contents in bulk soil and aggregates. Pearson correlation and path model analyses showed that the NO emission was positively correlated with soil carbon and nitrogen fractions and microbial carbon and nitrogen contents among organic and chemical fertilizer treatments. Long-term application of organic and chemical fertilizers strongly regulated NO emissions via affecting the distribution of carbon and nitrogen contents in soil fractions and changing microbial biomass and substrate availability. In conclusion, the application of organic fertilizers could significantly facilitate NO emission by increasing the available soil carbon and nitrogen pools as well as microbial carbon and nitrogen contents. The application of organic fertilizers mitigated the positive effects of chemical fertilizers on NO emissions. Appropriate amounts of organic fertilizers should be used when applying chemical fertilizers, in order to balance the comprehensive effects of fertility improvement with nitrogen loss and greenhouse gas emissions.