This study used a novel combination of cellulose nanocrystals (CNCs) and calcium oxide (CaO) nanocomposite (CaO/CNCs) for the production of biodiesel from waste cooking oil. The filter paper was used as a raw cellulose source to produce the CNCs from the acid hydrolysis of cellulose with sulfuric acid. The as-synthesized CaO/CNC nanocomposite is recyclable and environmentally friendly and was characterized using Fourier transform infrared spectroscopy, energy dispersive X-ray, scanning electron microscopy, and X-ray diffraction. The optimum process parameters investigated are a 20:1 methanol-to-oil molar ratio, 3-weight percent catalyst concentration, 60 °C temperature, and 90 min of reaction time. Under the optimum conditions, a biodiesel yield of 84% was obtained. The CaO/CNC nanocomposite achieved five times reusability, indicating its effectiveness and reusability in the transesterification reaction. The synthesized biodiesel chemical composition was examined using FTIR, GCMS, 1H-NMR, and 13C-NMR, and its properties, including specific gravity, color, flash point, cloud point, pour point, viscosity, sulfur content, sediments, water content, total acid number, cetane number, and corrosion test, were ascertained using ASTM standard practices. The outcomes were determined to fulfill global biodiesel standards (ASTM 951, 6751). Five successive transesterification processes were used to test the regeneration of the catalyst; the first three showed no distinct change, while the fifth cycle showed a reduction of up to 79%. The innovative composite CaO/CNC and used cooking oil are stable, affordable, and extremely successful for long-term biodiesel generation.