Micro-algae are sustainable alternative biofuel's biomass sources compared to a myriad of fossil fuel energy sources owing to their high triglycerides content, renewable nature, ability to thrive in unfavourable environments, high growth rate and carbon-capture and sequestration characteristics. In this present work, the effects of transesterification routes on the biofuel yield and quality (conversion efficiency), physical and chemical characteristics of Chlorella protothecoides micro-algae biodiesel were experimentally investigated through API, ASTM D6751-07b and EN 14,214 standard recommended techniques. The required heterotrophic micro-algae cells were cultured by the addition of 10 g/L of glucose (organic carbon source) to the standard agar medium in a 1000mL-corning Erlenmeyer flask with aseptic connector and with urea as nitrogen source. The extraction was done in a batch super critical fluid extraction apparatus equipped with a liquid CO2 pump. A high-performance liquid chromatography system coupled with an electro-spray ionization mass spectrometry equipment (HPLC-ESI-MS/MS) and UV light detector was developed for the rapid fatty acids' identification and quantification. While fatty acids methyl esters characterization was done by a Bruker FTIR Spectrometer with ATR sampling accessories. Similarly, the fuel's ignition and combustion qualities and oxidation stability were measured through cetane rating, heating value and induction periods experiments respectively. An oil yield of 68.7 % was obtained. This supports the efficiency of the SC-CO2 extraction process and the ability of heterotrophic growth to bring about increased biomass feed stock and higher accumulation of lipids (triglycerides) in its biomass. Preliminary results from HPLC/ESI/MS/MS analysis showed that the micro-algae oil consists essentially of long, straight chain unsaturated fatty acids (about 91 %) with 63.1 %,18.75 %, 6.8 % oleic, linoleic and linolenic components respectively and with cis-configuration. Whereas, palmitic (4.74 %), stearic (1.95 %) and arachidic (1.47 %) are the major saturated lipids. Finally, the SCM biofuel route is the most efficient in terms of ester yield (99.5 %) and quality, superior cold flow characteristics, higher cetane number and heating value, lower density and kinematic viscosity, negligible free and bound glycerol and better oxidative stability characteristics. This translates into superior performance, combustion and emission characteristics.