A new green microalga isolate Micractinium sp. GA001 was found to accumulate long-chain fatty acids, and the strain was subjected to flow cytometry-based adaptive evolution approach to produce improved phenotypes. At first, original phenotype of new isolate GA001 was well characterized followed by establishment of flow cytometry conditions in combination with fluorescent dyes BODIPY and Nile Red, to screen intracellular long-chain fatty acids in GA001. Fluorescent dyes staining and flow cytometry analysis revealed the progressive accumulation of desirable lipid components in GA001. Further, a flow cytometry-based strategy was used to selectively isolate and enrich particular GA001 phenotypes with higher accumulation of long-chain fatty acids, under nitrogen-depletion and -repletion conditions. This strategy yielded an improved population with high lipid content than original population. Micractinium sp. GA001 was proved to be a promising strain with improved phenotypes for the production of large-scale target-specific long-chain fatty acids.