Biodiesel from vegetable oil seed crops is the potential substitute for petro-diesel in the current era of every crisis. This article is confined to the transesterification of mustard oil (Brassica campestris L.) into biodiesel. The maximum biodiesel yield was 84% by using 2.5% alkali methoxide as the catalyst. The technical tools and processes used for monitoring of the transesterification reaction and characterization of biodiesel were GC-MS, H-1 NMR, (CNMR)-C-13, and FT-IR. The characteristic fuel properties of 100% mustard oil biodiesel, i.e., Kinematic viscosity @ 40 degrees C (ny) 5.775 Flash Point 110 degrees C, Pour Point -18 degrees C, Cloud Point -6.3 degrees C, Cetane number 53, and density @ 15 degrees C Kg/Lit 0.0871, were in accordance with ASTM standards. This study stated that mustard oil biodiesel is considered as a potential option for renewable energy, if mass cultivation is practiced through agriculture programs at global perspective.