One-time application of controlled-release blended fertilizer (CRBF, a mixture of five nitrogen (N) fertilizers in a certain ratio) can achieve high yield and N use efficiency (NUE) in rice (Oryza sativa L.). However, the effects of CRBF with one-time application on root spatial distribution and physiological characteristics remain unclear. We measured the effects of CRBF with one-time application on rice yield, NUE, root morphology and growth, and N uptake capacity in field and root box experiments. Six N treatments were set up: no nitrogen (N₀), high-yield three-split application of urea as a control (CK), urea (U) with broadcast, U with side-deep fertilization, CRBF with broadcast, and CRBF with side-deep fertilization. Our findings showed that root characters were positively correlated with yield and NUE. Compared to CK and U treatments, CRBF with one-time applications increased root characters (including root biomass, root N uptake, root activity, and the expression level of ammonium transporters) at tillering and heading stages. The root length, surface area and volume in the 0–10 cm soil layer enhanced under CRBF with one-time applications at tillering stage, and in the 0–20 cm soil layer at the heading stage. This contributed a 5.96%–39.40% and 3.69%–16.87% increase in plant dry matter accumulation and N uptake, and a 2.08%–18.28% and 14.60%–149.57% increase in yield and NUE, in 2022 and 2023, respectively. Taken together, our findings showed that one-time application of CRBF could increase rice yield and NUE by optimizing the root morphology distribution and N uptake.