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The amplification and cloning of promoters are regularly employed procedures to study the mechanism of gene regulation. In the present study we developed a method to construct T-vectors used for the direct and unidirectional cloning and analysis of promoters. These so-called T-vectors pEGFP-T and pGL3-T were derived from their parent promoterless vectors pEGFP-1 and pGL3-basic, respectively. To construct the T-vectors, an AhdI recognition site within the Ampr gene in pGL3-basic was silent mutated using overlap extension PCR. Then, a specially designed AhdI cassette was cloned into the respective parent vectors. The procedures of the T-vector construction involved a strategy to minimize the background of nonrecombinant transformants and to eliminate reverse orientation of the PCR products into the T-vectors. The cloning efficiencies of the two T-vectors were both above 85% when tested with a PCR product amplified from a sequence that was pre-confirmed to be able to initiate transcription, and moreover, the constructs harbored the inserts in a desired orientation at a >90% rate. In transient transfection assays, we demonstrated these T-vectors are functional. Thus, the present study provides an easy method to construct a series of T-vectors used for promoter characterization.