Abstract: The application of organic fertilizers promotes efficient chemical fertilizer use, but the effects and mechanisms of organic fertilizers with different maturation degrees are unresolved. To explore the effects of applying organic fertilizer with different maturity degrees on chemical nitrogen utilization efficiency for a practice of combined organic-inorganic fertilizers application, a pot experiment of lettuce (Lactuca sativa var. ramosa Hort.) was conducted using nitrogen-15 (¹⁵N) tracer technology. The organic fertilizers with different maturity degrees, which indicated by the germination index of cress (Lepidium sativum L.) (GI), were applied with ¹⁵N-labelled chemical fertilizer. A treatment without organic fertilizer application (CK) was set up as the control and following the principle of equal nutrient and carbon input, three treatments with different maturity degrees of organic fertilizer: ¹⁵NPK (nitrogen-15, phosphorus, potassium) + 50% GI organic fertilizer (GI50), ¹⁵NPK + 80% GI organic fertilizer (GI80), and ¹⁵NPK + 100% GI organic fertilizer (GI100) were tested. The results showed that, compared to CK, the GI50, GI80, and GI100 treatments significantly (P < 0.05) increased lettuce biomass, ¹⁵N uptake and ¹⁵N use efficiency by 30.5%-56.1%, 40.0%-91.0%, and 15.5%-41.8%, respectively. The biomass, ¹⁵N uptake, and ¹⁵N use efficiency of GI80 significantly (P < 0.05) increased by 17.1%, 31.8%, and 35.4%, respectively; those of GI100 significantly (P < 0.05) increased by 19.6%, 15.8%, and 22.8%, respectively, compared to GI50. Compared to CK, ammonium-nitrogen (¹⁵NH₄⁺-N) in GI50, GI80, and GI100 treatments significantly increased by 44.9%-74.2% (P < 0.05), nitrate-nitrogen(¹⁵NO₃^--N) significantly decreased by 8.4%-38.1% (P < 0.05), and net nitrification rate significantly decreased by 10.8%-24.6% (P < 0.05). Compared to GI50, GI80 increased ¹⁵NH₄⁺-N by 7.9%-11.5%, significantly decreased ¹⁵NO₃^--N and net nitrification rate by 18.5%-50.4% (P < 0.05) and 15.0%-28.2% (P < 0.05), respectively; GI100 significantly increased ¹⁵NH₄⁺-N by 11.5%-26.9% (P < 0.05), significantly decreased ¹⁵NO₃^--N and net nitrification rate by 15.8%-22.7% (P < 0.05) and 12.5%-23.9% (P < 0.05), respectively. The microbial biomass nitrogen (MBN) and MB¹⁵N in soil slowly increased. Compared to CK, GI80 and GI100 treatments significantly increased MB¹⁵N by 67.3%-94.1% (P < 0.05). Compared to GI50, GI80 and GI100 treatments increased MB¹⁵N by 6.0%-23.8% and 6.9%-25.5% (P < 0.05), respectively. The MB¹⁵N in each treatment accounted for 54.9%-71.6% (P < 0.05) of the MBN. Correlation analysis showed that MB¹⁵N and ¹⁵NH₄⁺-N had significant positive correlations with the ¹⁵N absorptive amount and ¹⁵N utilization rate; and the redundancy analysis showed that MB¹⁵N was the key driving factor of the absorption and utilization of ¹⁵N in chemical fertilizers. Therefore, an appropriate increase in the maturity of organic fertilizer (GI ≥ 80%) when applying organic and inorganic fertilizers can enhance the nitrogen fixation ability of soil microorganisms, improve soil nitrogen levels, slow the conversion rate of soil ¹⁵NH₄⁺-N to ¹⁵NO₃^--N, reduce the net nitrification rate, and inhibit soil nitrification, thereby improving the nitrogen utilization efficiency of chemical fertilizer. The results of this study provide an important basis for understanding the mechanisms of appropriately improving the maturity degree of organic matter to enhance chemical nitrogen use efficiency.