Nucleic acid testing technology has made considerable progress in the last few years. However, there are still many challenges in the clinical application of multiple nucleic acid assays, such as how to ensure accurate results, increase speed and decrease cost. Herein, a three-way junction structure has been introduced to specifically translate analytes of loop-mediated isothermal amplification to a catalytic hairpin assembly. For different analyses, a well-optimized nucleic acid circuit can be directly applied to detection, through only one-component replacement, which only not avoids duplicate sequence design but also saves detection cost. Thanks to this design, multiple and logical analysis can be easily realized in a single reaction with ultra-high sensitivity and selectivity. In this paper, Mycoplasma pneumoniae and Streptococcus pneumoniae can be clearly distinguished from the clinical mixed sample with negative control or one analyte in one tube single fluorescence channel. The fair experimental results of actual clinical samples provide a strong support for the possibility of clinical application of this methodology.