This study developed a new approach to produce sterile, hyperpolarized [¹³C,¹⁵N₂]urea as a novel molecular imaging probe and applied it for first-ever healthy brain volunteer studies. Hyperpolarized [¹³C,¹⁵N₂]urea, as a small, metabolically inert molecule, offers significant advantages for perfusion imaging due to its endogenous nature and excellent safety profile. The developed methods achieved a hyperpolarized [¹³C,¹⁵N₂]urea solution (132 ± 6 mM) with 27.4 ± 5.6% polarization and a T₁ = 50.4 ± 0.2 s. In healthy brain volunteer studies, high-resolution ¹³C imaging captured blood flow with a spatial resolution of 7.76 × 7.76 × 15 (or 10)mm³ over ~1 min following hyperpolarized [¹³C,¹⁵N₂] urea injection, visualizing detailed vascular structures. Time-to-peak and centroid analyses showed consistent arterial and venous signal patterns across subjects. Findings suggest hyperpolarized [¹³C,¹⁵N₂]urea may have applications beyond brain imaging, including the non-invasive perfusion assessment in various organs, cancer microenvironment, and renal function, paving the way for clinical translation.