Biodiesel production from cotton seed oil and its characterization is focussed in this experimental investigation. As the free fatty acid content is less than 2%, base catalysed transesterification process is adopted with methanol and potassium hydroxide as solvents. The transesterification process is optimized by varying the methanol to oil molar ratio and catalyst concentration with standard operating parameters. By this process, 93% of biodiesel is obtained. The biodiesel is subjected to gas chromatography-mass spectrometry analysis which revealed the presence of linoleic acid as the major constituent. Fourier transforms infra-red and nuclear magnetic resonance spectral studies attributed the gas chromatography-mass spectrometry data confirming the presence of fatty acid methyl esters.