Due to the excellent activity of trans-cleavage and target recognition, the recently discovered CRISPR/Cas12a systems provide a promising opportunity for designing fluorescence biosensing. In the reported CRISPR/Cas12a-based biosensing platform, TaqMan probe is widely used as the reporter. However, the TaqMan probe provides single-wavelength fluorescence changes, which is easily influenced by various analyte-independent confounding factors to produce false-positive signals. In this study, a ratiometric fluorescent probe was designed to act as the reporter of a CRISPR/Cas12a-based system. As a proof-of-concept, fluorescein (FAM) and tetramethylrhodamine (TAMRA) were chosen as the two fluorescence dyes to label one short ssDNA at 5' and 3' ends, respectively, which was designed as one ratiometric fluorescent probe. When the ratiometric probe excites at 480 nm, duo to FRET effect, the probe emitted the 580 nm-fluorescence of TAMRA. The activated Cas12a can cleave the dual-labeled ssDNA, resulting in a decrease in the TAMRA's fluorescence and an increase in the FAM's fluorescence. This dual-response fluorescent probe can act as the reporter of the CRISPR/Cas12a-based biosensing platform. Compared with the classic TaqMan, CRISPR/Cas12a-based biosensing with the ratiometric probe as the reporter not only exhibited higher sensitivity but also could distinguish and avoid false positive signals.