Poultry manure, which contains abundant water‐soluble organic matter (WSOM), is commonly applied in plastic greenhouse vegetable production. There is little research on whether WSOM stimulates nitrous oxide (N2O) emission after poultry manure application. A 30 day incubation experiment and a field experiment are conducted to investigate the response of the N2O flux after poultry manure or WSOM inputs. The cumulative N2O emissions from soil amended with WSOM in the incubation experiment are two to four times than those from unamended soil. Similarly, in the field experiment, the N2O emissions significantly increased by 225% (to 1.13 kg N ha−1) after poultry manure and irrigation application compared to a control during summer fallow season, accounting for ≈20% of the annual N2O emissions from plastic greenhouse vegetable production. In conclusion, WSOM in poultry manure increases N2O emissions from plastic greenhouse soil during its initial application; moreover, N2O emissions during summer fallow season should be included in the N2O emission inventory of plastic greenhouse vegetable production. Measures for controlling N2O emissions such as avoiding nitrate accumulation and improving water and poultry manure management should be adopted in plastic greenhouse vegetable production.