A rapid and accurate detection method is required for variety identification in basic research, germplasm preservation, and field applications. The microalgae Nannochloropsis sp. and Chlorella sp. exhibit striking similarities in appearance, yet differ in nutritional composition. Nannochloropsis sp. is rich in eicosapentaenoic acid (EPA), while Chlorella sp. is not. EPA is very important to the breeding rate of aquatic seedlings. Using the wrong algae to nourish the bait organisms and feed the seedlings may lead to a decrease in the breeding rate. Visual identification of algae may result in misjudgments, and traditional methods such as chlorophyll, fatty acids, and gene sequencing take 2-5 days and are cost-intensive. In this study, a species-specific primer was designed based on the differences in the 18S ribosomal RNA (18S rRNA) gene sequences between Nannochloropsis sp. and Chlorella sp. The results indicate that using the species-specific primer NSCS 1 in polymerase chain reaction (PCR) enables the identification of Nannochloropsis sp. and Chlorella sp., as well as cross-contaminated samples, within 3 hours based on the different sizes of the amplified fragments. This helps avoid the negative impact on aquaculture outcomes due to the inadvertent use of the wrong algal species, contributing to the development of bait organisms in the aquaculture industry and the stable production of aquatic seedlings.