Many studies concerning breeding maize varieties are in progress in Korea and other countries. Double haploid technology is widely used for the development of commercial maize varieties worldwide, and has also been utilized in Korea since its introduction by the Maize Research Institute, Gangwondo. We performed a study to improve the efficiency of selecting haploid maize seeds using fluorescence imaging. It was verified that anthocyanin produced by the expression of R1-nj gene can be detected by fluorescence imaging, and we developed a high-throughput method for discriminating between haploid and diploid seeds. Compared with discriminating with naked eye, this method reduced the time for discriminating haploid and diploid maize by 91.7% and increased selection accuracy by 16.8% for haploid and 2.2% for diploid maize. This method enabled the acquisition of more haploid seeds and improved the efficiency of breeding research by shortening the time involved.