Recent advances in fluorescent biosensors have stimulated the development of molecular detection. We herein developed a new orthogonal logic operation of fluorescent biosensor with cell-free to accomplish the detection of atrazine (ATZ) and tetrachlorobiphenyls (PCB77). The transcriptional process to generate fluorescent RNA aptamers (Mango) was controlled by molecules-probe bindings, which regulate split T7 promoter transcription switches ON or OFF. Leveraging the rapid in vitro T7 transcription process and high signal-to-background ratio of the Mango-TO1-Biotin complex, this biosensor demonstrates remarkable sensitivity in detecting ATZ and PCB77, with detection limit of 1.56 pM and 10.2 pM. Moreover, the orthogonality of four logic gates (AND, NOR, INHIBT, NIMPLY) were utilized the ATZ and PCB77 as input to construct, which could be activated by utilizing the target probe-driven association. The output of the fluorescence signal was controlled by split/intact fluorescent RNA aptamer (Mango) to achieve flexible and sensitive orthogonal operations. Significantly, the development of two-input logic gates has enabled the modular detection of various small molecules, offering a promising approach to intelligent multi-input analysis. Predictably, with the advantages of sensitivity, flexibility and easy-to-operate, this orthogonal logic gate platform holds immense potential in small molecules detection.