The 10–23 DNA enzyme (10–23 DNAzyme), a single-stranded DNA (ssDNA) molecule, can efficiently and specifically cleave almost any target RNA molecules. Therefore, it is regarded as one of the promising tools in gene therapy. However, there are still some obstacles, such as low efficiency of cellular uptake and instability in vivo, in its application. Taking advantage of the mechanism of Moloney mouse leukemia virus (MMLV) reverse transcriptase (RT), we investigate the construction of a novel ssDNA expression vector in this study. In order to improve the expression efficiency, the mmlv-rt gene and ODN-PMT (an oligodeoxynucleotide including other essential sequences for generating ssDNA) were cloned into a single plasmid under the control of 2 separated promoters. The ability of the vector to generate specific 10–23 DNAzyme in mammalian cell was tested by constructing a tryptophan–aspartate-containing coat protein (taco) gene-specific 10–23 DNAzyme expression plasmid. The potential of the expressed 10–23 DNAzyme to suppress TACO expression was also investigated. Our results indicated that this vector generates desired 10–23 DNAzyme in mammalian cells. The expressed 10–23 DNAzyme targeting taco gene can reduce TACO expression both at mRNA level (by 78.26%) and at protein level (by 75.30%).