Clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated proteins (Cas)-based detection platform opens up a new avenue for biosensing owing to its programmability, sequence specificity, and high-base resolution upon nucleic acid targets. In the current study, we utilized the target DNA-induced non-specific single-stranded DNA cutting activity (trans-cleavage) of CRISPR-Cas12a to fabricate an aptamerAptamers-mediated fluorescent biosensor for sensitive and selective detection of adenosine triphosphate (ATP). In this sensor, we designed an ATP-binding aptamerAptamers as the target ssDNA for Cas12a; thereby, in the presence of ATP, the ATP-binding aptamerAptamers would be occupied by ATP, and less target ssDNA can be processed by Cas12a, which gave rise to an ATP concentration-dependent change in fluorescence resulted from trans-cleavage of doubly labeled ssDNA reporter. In this way, we successfully “translated” ATP signals to nucleic acid signals that can be amplified by Cas12a process. The dynamic detection range was from 1 μM to 200 μM, and the limit of detection was 400 nM. The entire sample-to-answer time for this biosensor was around 40 min. Overall, this novel biosensor balanced sensitivity, specificity, detection time, and ease of use. Our proposed biosensor provided a principle-of-proof for detecting small molecules by using CRISPR-Cas12a system, which would unlock its potential and further its futuristic applications in the field of diagnostics.