The subtraction method is a quick and economical technique to scan differential gene expression. However, most subtraction methods are limited by the complexity and length of cDNA samples. To overcome this problem, we developed a novel method to identify the unique full-length cDNAs in two complicated tissue or cell types. This method, duplex-specific nuclease (DSN)-mediated transcriptome subtraction (DTS), is based on the normalization strategy of the crab duplex-specific nuclease and the subtraction method of suppression subtractive hybridization. DSN eliminates nearly all of the common sequences in the tester and driver cDNA samples after the first hybridization step, ensures accurate discrimination between the tester and the driver cDNA samples, and enriches the full-length differential cDNAs from the tester. Using the DTS method, we have successfully identified an 1812-bp additional GUS gene from the complicated Arabidopsis seedling cDNA library. We also employed DTS to detect the differences in mRNA expression of salt-treated Arabidopsis seedlings to illustrate further the efficiency of the subtraction method.