Abstract: 5 fatty acyl desaturase (Fad) is the key enzyme for the biosynthesis of long chain polyunsaturated fatty acids. Abalone, Haliotis discus hannai Ino, has two genes of 5 Fad (Hdhfad1 and Hdhfad2), of which the cDNA sequences are highly similar (96.82 %). In this study, an efficient Real-Time quantitative RCR (RT-qPCR) assay based on the 2-Ct method was established for measuring the expression levels of Hdhfad1 and Hdhfad2. Data were normalized to the gene expression levels of -actin and ribosomal protein S9 (RPS9). The effects of different dietary lipids on the expressions of Hdhfad1 and Hdhfad2 were studied in muscle tissue of abalone using this assay. We prepared three types of diets that contain different dietary lipids－tripalmitin (TP diet), ARA oil (AO diet) and EPA oil (EO diet). The results showed that primers designed for Hdhfad1, Hdhfad2, -actin and RPS9 were specific, with the amplifying efficiency of 1.05, 0.99, 0.97 and 0.98 respectively. The efficiency of these primers was suitable for the 2-Ct method. The optimal annealing temperature and reaction volume for the RT-qPCR amplification were 52℃ and 25 L respectively. We concluded that this assay was rapid and sensitive in analyzing the expressions of 5 Fad in abalone. This assay demonstrated that the expression levels of Hdhfad1 and Hdhfad2 were significantly decreased in abalone fed with EO or AO diet compared to those of TP group.