Biodiesel is an alternate renewable, biodegradable, non-toxic fuel similar to conventional fossil fuel. It is usually produced from vegetable oil, animal fat, tallow, non-edible plant oil and waste cooking oil. Residue oil components and by-products from the production process or contamination during handling and storage could affect the quality of the biodiesel. The molecular compositions of biodiesel samples have been investigated by a combination of NMR spectroscopic methods. The use of NMR spectroscopy is a novel method to biodiesel characterisation is implemented to fully characterise and assign the molecular structure of biodiesel samples and to identify and quantify the moieties of the molecules, particularly the unsaturated long-chain alkyl esters. The NMR spectroscopic method was also implemented to evaluate the transesterification process; the amount of trans-esterified biodiesel in the samples and the amounts of un-reacted different types of glycerides. Furthermore, the NMR spectroscopic method is developed to quantify methanol in biodiesel and proposed here as alternative to the official method.