A high-yield method was developed for producing a TA-cloning vector suitable for blue/white colony selection from a unique parent plasmid containing a dual lacZ gene system through a one-step restriction enzyme digestion, which creates a single-base 3'-overhang. The dual lacZ gene system was realized by inserting an inner lacZ gene between two single-base 3'-overhangs, creating restriction enzyme sites within the reading-frame-adjusted outer lacZ gene sequence in the parent plasmid. The proposed method overcomes problems, such as the inefficient digestion frequently observed when generating a TA-cloning vector and the difficulty of purifying TA-cloning vectors from the digestion mixture, while maintaining the applicability of blue/white colony selection. Moreover, the use of TA-cloning vector prepared by the proposed method can provide the distinguish tool of transformants carrying the cloning product from those carrying contaminating parent plasmids, recircularized plasmids derived from incompletely digested parent plasmid fragments, or intra-molecularly ligated TA-cloning vectors derived from T-overhangs missing TA-cloning vectors (instability of the T-overhangs is another important consideration when designing TA-cloning vectors) by making all colonies except those carrying the cloning product appear blue during blue/white colony selection.