This study explored the effects of combined application of straw and cattle manure on the activation of soil acidolytic nitrogen fractions and greenhouse gas emissions under different initial nitrogen levels. An indoor incubation experiment was conducted with two soil background levels (low nitrogen, LN; high nitrogen, HN). Under equal carbon input, six organic material ratios (S0M0, S100M0, S75M25, S50M50, S25M75, S0M100) were set up, totaling 12 treatments. Soil carbon fractions, acidolytic nitrogen fractions, microbial biomass, enzyme activities, and emissions of CO2 and N2O were determined.The results showed that organic material addition significantly increased soil total organic carbon and particulate organic carbon, with higher increments in LN soil (6.7% and 52.7%) than in HN soil (4.9% and 18.6%). Total acidolytic nitrogen, amino acid nitrogen and ammonium nitrogen peaked in sole cattle manure treatment. The increases relative to control in LN soil (9.2%, 14.9%, 12.2%) were slightly lower than those in HN soil (9.2%, 16.1%, 11.9%). Microbial biomass nitrogen was highly sensitive to cattle manure input; high manure ratios increased it by 38.7%~48.6% in LN soil and 81.9%~104.0% in HN soil. Microbial biomass C/N ratio declined with the increasing proportion of cattle manure. Microbial nitrogen limitation existed in all treatments. Cattle manure addition alleviated carbon limitation but aggravated nitrogen limitation under high nitrogen level. Cumulative CO2 emissions decreased with increasing cattle manure proportion and were significantly lower in HN soil. N2O emissions rose markedly with elevated nitrogen level and cattle manure ratio, with the highest value in mixed treatments. Correlation analysis revealed that microbial biomass nitrogen was positively correlated with ammonium nitrogen and leucine aminopeptidase activity, while N2O emission was significantly positively related to amino acid nitrogen, ammonium nitrogen and particulate organic carbon. In conclusion, combined organic application optimizes soil carbon and nitrogen pool structure, which is regulated by initial soil nitrogen level. High-proportion cattle manure is recommended for low-nitrogen soil to synchronize nitrogen activation, carbon sequestration and emission reduction, while nitrogen loss risk should be concerned in high nitrogen soil.