The present work investigates the production of biodiesel from Euglena sanguinea microalgal bio-oil using calcium methoxide as a heterogeneous catalyst. The catalyst was synthesized and characterized by Fourier Transform Infra-red (FTIR) spectroscopy, Scanning Electron Microscopy (SEM), X-ray Diffraction (XRD), Brunauer-Emmett-Teller (BET), basicity, and basic site strength analysis. Initially, bio-oil was extracted from mass-cultivated biomass obtained from Euglena sanguinea algae. It was further pretreated and transesterified using calcium methoxide catalyst at various experimental conditions by which an optimum yield of 94.83% was achieved. The catalyst yielded above 90% up to 5 cycles of recovery and recycling. The kinetic studies were investigated at various reaction temperatures to find the rate of reaction. The activation energy and pre-exponential factor for the transesterification reaction were found to be 99.33 kJ mol(-1) and 1.07 x 10(14) min(-1) respectively. The properties of the produced biodiesel were within the limits of ASTM D6751 standard.