This study is reporting the biofuel synthesis and characterization from the novel nonedible feedstock cocklebur seeds oil. The Cocklebur crop seeds oil was studied as a potential source for biofuel production based on the chemical, structural and fuel properties analysis. The oil expression and FFAs content in cocklebur crop was reported 37.2% and 0.47 gram KOH/g, using soxhlet apparatus and acid base titration method, respectively. The maximum conversion and yield of the cocklebur crop seeds non-edible oil to biofuel was pursued 93.33%, using transesterification process. The optimum protocol for maximum conversion yield was adjusted: 1:7 oil-methanol molar ratios, ZnO nano-particle concentration 0.2 gm (w/w), reaction temperature 60 • C, and reaction time 45 min, respectively. ZnO nano-particle was prepared by a modified sol-gel method, using gelatin and the particle was XRD, TEM, XPS, and UV-vis spectroscopies. Qualitatively, the cocklebur crop synthesized biofuel was quantified and structurally characterized by GC/MS, FT-IR, NMR, and AAS spectroscopies. Quantitatively, the fuel properties of cocklebur crop biofuel was analyzed and compared with the international ASTM and EN standards.