In recent times, the world has been confronted with an energy crises due to the depletion of fossil fuel resources, increased petroleum prices, and great environmental concerns. The situation has led to the search for an alternative, sustainable and clean fuel from vegetable oils and their derivatives. This article reports an optimized protocol for the production of biodiesel from a new source of non edible hemp oil (Cannabis sativa L.) through base catalyzed transesterification. The hemp oil is investigated for the first time in this study as a potential source of biodiesel based on detailed physico-chemical analysis. The quantity and quality of hemp oil biodiesel (HOB) is evaluated through fuel properties analysis and GC-MS, FT-IR and NMR techniques. The free fatty acid number of crude hemp oil was 0.98 mg/g and 2.11 mg/g against NaOH and KOH catalysts. The percentage of conversion of crude oil to fatty acid methyl esters (FAME) was 90% at 6:1 molar ratio (Methanol:Oil) by using 20% NaOH catalyst at 60 degrees C. The most important variables affecting the FAME yield during transesterification are reaction time, catalyst concentration, molar ratio and reaction temperature. The HOB was found to be clean, environmentally friendly, and exhibit fuel properties within the range of American Standard for Testing Material.