This work describes a highly sensitive and rapid amperometric biosensor for organophosphate compounds (OPs) based on immobilization of acetylcholinesterase (AChE) on a novel TiO2-decorated graphene (TiO2-G) nanohybrid, which was constructed by in situgrowth of TiO2 nanoparticles (NPs) on the graphene sheet. The well-dispersed TiO2 NPs eliminated the restacking of TiO2-G nanohybrids. Due to the integrating of TiO2-G nanohybrids, the as-prepared biosensor showed high affinity to acetylthiocholine (ATCl) with a Michaelis–Menten constant (Km) value of 0.22 mM, and rapid inhibition time (3 min). Further, based on the inhibition of OPs on the enzymatic activity of the immobilized AChE, and using carbaryl as a model compound, the inhibition of carbaryl was proportional to its concentration ranging from 0.001 to 0.015 and 0.015 to 2 μg mL−1 with a detection limit of 0.3 ng mL−1 (S/N = 3). The developed biosensor exhibited a good performance for organophosphate pesticide detection, including good reproducibility and acceptable stability, which provided a new and promising tool for the analysis of enzyme inhibitors.