Biodiesel derived from microalgae is a sustainable, renewable, and readily biodegradable alternative fuel. The use of γ-irradiation to promote biological processes in microalgae has significantly increased in recent years. The purpose of the study was to evaluate the effects of γ-irradiation in the characterization and production of microalgae biodiesel from Chlorella vulgaris grown in BG-11 media utilizing many analytical methods, including elemental analysis (CHNSO), fourier-transform infrared spectroscopy (FTIR) and gas chromatography–mass spectrometry (GC–MS). The data proved that γ-irradiation  treatments (200 Gy) can be used for enhancing the amount of particular fatty acids in C. vulgaris that influence the quality of biodiesel in order to meet international biodiesel standards. A total of twelve different fatty acids were found. Furthermore, the majority of the unsaturated and saturated fatty acids in fatty acid methyl esters (FAMEs) have carbon chains that range from C12 to C24. C. vulgaris is the most promising source of biodiesel according to the data since it contains a cetane number (88.6), a low iodine value (103.73), a high content of saturated fatty acids (87.2%), and numerous other biodiesel characteristics that are within the acceptable range. γ-irradiated C. vulgaris has higher elemental levels of produced blended biodiesel (CHNO), as compared to the control. Sulphur was not present in the produced biodiesel from C. vulgaris. The increased oxygen level of bio-oil makes it desirable for use in transportation fuel manufacturing. The proportion of carbon increases gradually, improving combustion efficiency and encouraging full combustion of fuels based on carbon.