Abstract: After industrialization occurred, fossil fuel reserves decreased drastically, and environmental pollution caused by fossil fuel consumption became more serious. Thus, clean and renewable energy alternatives have recently attracted increasing amounts of attention. Microalgae have many advantages, such as a high growth rate, suitable fatty acid profiles and high lipid content, which makes them one of the most promising sources for renewable energy production. However, making a profit from biodiesel production based on microalgae is difficult because of the high cost. There are various strategies to reduce the cost of biodiesel production, such as breeding the best microalgae strains through chemical mutagenesis, which is one of the most important methods. Although this method is effective, extraordinarily heavy follow-up work impedes its promotion and application. Fortunately, flow cytometry and lipophilic fluorescent dyes (BODIPY^505/515) provide the opportunity to screen for the best mutants using high throughput. In this study, multiple mutants of Nannochloropsis oceanica were obtained by using chemical mutation (nitrosoguanidine, NTG). We developed a method based on flow cytometry and BODIPY^505/515, which can screen microalgal candidates using high throughput. Our results showed that the optimal concentration and staining time for BODIPY^505/515 were 0.87 μg/mL and 10min, respectively. Based on this method, four outstanding microalgal strains were obtained. To verify the reliability of the screening method, we further investigated the performance of these four strains cultivated in a column photobioreactor. The results showed that lipid productivity of the four mutants was remarkably higher than that of the wild type, and the highest value was 27.32 mg/(L·d), demonstrating that this method is effective and feasible. To further evaluate the potential of candidates for biodiesel production, we investigated factors such as the fatty acid profiles, chlorophyll a, and PSII quantum efficiency. The results indicated that all of these four mutants possessed great potential for biodiesel production.