To achieve efficient genetic transformation in Dunaliella viridis, this study optimized key parameters including culture media, antibiotic selection markers, and electroporation conditions. The results demonstrated that adding 0.5% agar to solid media enabled normal algal growth. The D. viridis strain showed insensitivity to glufosinate-ammonium but was susceptible to chloramphenicol and chlorate, with growth significantly inhibited by 400 μg/mL chloramphenicol in liquid media and 300 μg/mL in solid plates. For electroporation, cells were harvested by centrifugation at 1 700g for 3 min to achieve optimal cell collection efficiency. Using the NR gene knockout vector pKS-diaCas9-sgRNA-NRKO delivered via electroporation, nitrate reductase (NR)-deficient mutants were successfully selected on 100 mmol/L (30 g/L) sodium chlorate plate. Sequencing confirmed precise knockout at the second exon of the NR gene. This work establishes a foundational protocol for employing D. viridis as a bioreactor in genetic engineering applications.