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The current study focuses on the synthesis of Cerium oxide (CeO₂) nanocatalyst via Tragacanth Gum (TG) using the wet impregnation method and its application for sustainable biodiesel production from a novel, non-edible <i>Descurainia sophia</i> (L.) Webb ex Prantl seed oil. The <i>D. sophia</i> seed oil has higher oil content (36 wt%) and free fatty acid (FFA) value (0.6 mg KOH/g). Innovative analytical methods, such as X-ray diffraction (XRD), scanning electron microscopy (SEM), energy dispersive X-ray (EDX), transmission electron microscopy (TEM), thermogravimetric analysis (TGA), and Fourier transform infrared spectroscopy, were used to characterize the newly synthesized, environmentally friendly, and recyclable CeO₂-TG phytonanocatalyst (FT-IR). The results show that the CeO₂-TG phytonanocatalyst was 22 nm in diameter with a spherical shape outer morphology, while the inner structure was hexagonal. Due to low FFA content, the <i>D. sophia</i> seed oil was pretreated and transesterified via a single step. Using varying parameters, the optimized process variables were determined via Response Surface Methodology (RSM). The optimum process values were 8:1 methanol to oil molar ratio, 0.3 wt% catalyst concentration, 90 °C temperature, and reaction time of 210 min with 98% biodiesel yield. The recently created phytonanocatalyst was reliable and effective, with three times reusability in the transesterification reaction. Thin layer chromatography (TLC), FT-IR, gas chromatography–mass spectroscopy (GCMS), and Nuclear magnetic resonance (NMR) analyses were used to characterize the synthesized biodiesel. Physico-chemical properties of <i>D. sophia</i> biodiesel, i.e., Kinematic viscosity (4.23 mm²/s), density (0.800 kg/m³), pour point (−7 °C), cloud point (−12 °C), and flash point (73.5 °C) agree well with international biodiesel standards (ASTM-6751, 951), (EU-14214), and China (GB/T 20828) standards. The results show that the synthesized nanocatalyst demonstrated remarkable stability, indicating a bright future for industrial biodiesel production from low-cost feedstock.