BNI (biological nitrification inhibition) derived from root exudates is typically used in rotation cropping systems to increase the crop nitrogen efficiency (NUE). Recently, the root exudates secreted by three sugarcane varieties in Thailand have been found to inhibit the nitrification and accumulation. The objective of the study was to evaluate the effectiveness of the sugarcane root exudate on (i) soil nitrification rate and N dynamics (soil mineral N and nitrous oxide (N2O) emission) and (ii) growth, yield, and 15N recovery by soybean under greenhouse conditions. The experiment was laid out as a randomized complete block design (RCBD), with five treatments and four replications: (i) N fertilizer (Control); (ii) Control + Uthong 13 sugarcane root exudate (UT13); (iii) Control + Khon Kaen 3 sugarcane root exudate (KK3); (iv) Control + wild cane sugarcane root exudate (Spone) and (v) Control + dicyandiamide (DCD). 15N fertilizer was applied to a micro-plot for 15N recovery. Soybean was planted and fertilized at 75 kg ha−1 N as (NH4)2SO4. Soil samples were collected at 1, 3, 7, 14, 21 and 28 days after N fertilization (DANF) for pH, potential nitrification rate (PNR), NH4+, NO3− and MBN measurement at 0–15 and 15–30 cm soil depths. A gas sample was collected by the soil chamber technique for an N2O emissions study on the soil sampling date. The soybean growth was monitored at 30 and 60 DAP (days after planting) for height, leaf area, leaf area index (LAI), SCMR and dry weight (DW). At soybean harvest, seed yield, yield components and N uptake were recorded. Our study revealed that the sugarcane root exudates significantly decreased PNR up to 14 DANF and increased NH4+ content in soil and reduced commutative N2O–N fluxes after 14 DANF, compared to N treatment (p < 005). The root exudate treatments not only increased growth (total DW and LAI at 60 DAP), yield (number of pods and seeds per plant) and N uptake (17–19%) but also 15N recovery in the total plant. This confirms the capacity of BNIs from sugarcane root exudates. In conclusion, sugarcane root exudates can effectively mitigate N2O emissions and enhance soybean N uptake under greenhouse conditions.