The present research was mainly focused on the production of biodiesel from Annona squamosa oil using a synthesized Ni-doped CaO nanocatalyst. The optimization of the transesterification reaction parameters was studied through response surface methodology. The highest biodiesel yield of 99.1% was achieved with the optimized conditions of 7.86% catalyst concentration, 442 RPM, 15.19:1 molar ratio of methanol to oil, reaction temperature of 55.8°C and reaction time of 63.3 min. The results obtained from reaction kinetics study showed a good fit with a first-order kinetic model. The activation energy and R2value were determined to be 53.7 kJ/mol and 0.90, respectively. The synthesized Ni-doped CaO nanocatalyst was characterized using Scanning Electron Microscope with Energy Dispersive X-ray Spectroscopy which confirms the presence of nickel, calcium and oxygen. Also, the average size of the nanocatalyst was found to be 48.79 nm. The Fourier Transform–Infrared Spectroscopy results showed the occurrence of functional groups such as C-H and C = O bonds in the synthesized Ni-doped CaO nanocatalyst. The presence of fatty acid methyl esters in the produced biodiesel was analyzed through Gas Chromatography-Mass Spectrometry analysis. The obtained results from the current study provides the possibility and insights for sustainable biodiesel production and a greener environment.