Although biothiols, including cysteine (Cys), glutathione (GSH), and homocysteine (Hcy) can be used to diagnose many diseases and research physiological metabolism in many physiological processes, in situ real-time detection and differentiation of biothiols is still challenging because their similar chemical properties and molecular structures. Herein, we utilized the native chemical ligation (NCL) reaction mechanism to develop a Förster resonance energy transfer (FRET) strategy for designing a cell penetration peptide TAT-modified ratiometric two-photon biothiols probe (TAT-probe). The TAT-probe can not only rapidly enter into mitochondria assisted by TAT peptide, but also simultaneously detect biothiols and sequentially distinguish GSH. When the TAT-probe was excited with 404/820 nm wavelength light, it showed a change in the ratio of fluorescence after adding biothiols, including a quenched red fluorescence intensity (λem = 585 nm) and an enhanced signal in green fluorescence intensity (λem = 520 nm). Excitingly, the TAT-probe excited at 545 nm could display a red fluorescence (λem = 585 nm) towards GSH and a quenched signal towards Hcy or Cys. This specific fluorescence response indicated the TAT-probe could effectively detect biothiols and differentiate GSH from Cys/Hcy in mitochondria. This work pioneered a new approach to design and synthesize biothiol-probes based on peptides and NCL reaction mechanism.