We identified a novel 12 bp promoter that significantly increased transcription efficiency. Unlike the standard 20 bp promoter, which contains both recognition and initiation regions, the new promoter contains only a recognition region and is more suitable for diagnostic applications due to its smaller size. This promoter effectively produced different light-up RNA aptamers via transcription. Moreover, we used the promoter to analyze RNase H activity and achieved a detection limit of 0.009 U mL⁻¹, which was significantly better than that achieved via previous methods. We propose that the new promoter may serve as a key component in various diagnostic applications.