Conventional nitrogen fertilizers are often constrained by a short effective period, high nitrogen loss rates, and low nitrogen use efficiency (NUE). Novel amino acid-based fertilizers, such as polyaspartic acid (PASP)-urea, offer a promising solution to mitigate nitrogen losses and enhance crop NUE. To evaluate the efficacy of PASP-urea in paddy fields, field and pot experiments were conducted in 2019 and 2020 using two rice cultivars, Shuhui 498 and Duohui 1. The study assessed three fertilizer treatments: PASP-urea, conventional urea, and a no-urea control. By analyzing soil and plant nitrogen content, root architecture, gene expression, and grain yield, this study investigated the relationship between PASP-urea application, soil nitrate distribution, and root architecture configuration. Compared with conventional urea, PASP-urea significantly increased nitrate concentration in the 0–10 cm soil layer for both rice varieties by 45.8–81.4% at 30 days after tillering fertilization. Concurrently, it significantly upregulated the expression of nitrate transporters NRT1.1A and NRT1.1B and promoted greater root distribution in the nitrate-enriched topsoil. This root distribution pattern promotes the uptake and utilization of nitrogen by rice plants. Consequently, PASP-urea increased grain yield by 6.7–11.7%, while nitrogen agronomic efficiency (NAE), nitrogen recovery efficiency (NRE), and nitrogen partial factor productivity (PFP) increased by 15.2–25%, 17.8–49.6%, and 6.8–11.2%, respectively. These results indicate that PASP-urea enhances NUE and is associated with increased topsoil nitrate concentration, upregulated nitrate uptake genes, and a remodeled root architecture, offering a viable strategy for sustainable rice production.