Arthrospira platensis (A. platensis) is an economically important microalgae because it has carbohydrates, lipids, proteins and a number of phytochemicals. It is also a valuable source used in the production of biodiesel and functional foods. In this study, A. platensis was exposed to electron beam irradation (240 kGy) and induced random mutagenesis for strain improvement. Several mutants were obtained, and the resulting mutant was designated as EB29. The growth rate and chlorophyll content of EB29 was similar to those of wild type. However, the lipid content of EB29 was increased seven-fold compared to that of wild type when comparing the nile red fluorescent intensity. Semi-quantitative analysis of EB29 using the calibration plot of standard lipid, triolein, represented 78.6 μg/mL, which increased 2 times compared to wild typ (41.4 μg/mL). When analyzing the fatty acid profile of EB29, polyunsaturated fatty acids (PUFAs), such as gamma-linolenic acid (GLA) in EB29 increased about six-fold. Moreover, fatty acids affecting the quality of biodiesel increased compared to that of wild type. Thus, electron beam could be used for the strain improvement of microalgae in order to accumulate PUFAs and alteration of fatty acid profile for biodiesel.