Recently, the ratiometric analysis of AChE activity based on the intrinsic dual-emissive fluorescent (FL) nanomaterials is urgently required. The AChE activity can serve as a promising biomarker of neuro-toxicity induced by organophosphorus pesticides. In this work, oxidized carbon dots (ox-CDs) with intrinsic dual FL peaks at 650 and 680 nm are obtained. Moreover, the FL peak at 680 nm can be effectively quenched by Ag + or Hg 2+ while that at 650 nm can be served as self-reference signal. The introduction of AChE and acetylthiocholine (ATCh) contribute to the generation of TCh, which can capture the metal ions via competitive association to recover their FL signal to the initial status. Furthermore, the AChE activity can be determined by the ratio of FL intensity at 680 nm to that at 650 nm. As for the ox-CDs-Ag + system, the linear range is determined to be 0.5-10 mU/mL and LOD is 0.127 mU/mL, while those for ox-CDs-Hg 2+ system are 0.05-1.6 mU/mL and 0.019 mU/mL, respectively. Furthermore, the fluctuation of plasma and cerebrospinal fluid (CSF) AChE activity in dichlorvos-poisoned and pralidoxime therapeutic rats has been monitored by the ox-CDs-Ag + system, which is essential for early diagnostics and prognostics of dichlorvos triggered neurotoxicity. • The activity of AChE is detected by intrinsic dual emissive CDs with ratiometric model • Great detection performance can be realized, in the aspects of lower LOD and faster response time • The fluctuation of AChE activity in OP intoxication and therapy can be monitored