This work reports a sensitive amperometric biosensor for organophosphate pesticides (OPs) fabricated through modifying glassy carbon electrode with acetylcholinesterase (AChE) immobilized on porous-reduced graphene oxide (pRGO). The pRGO sheets can not only provide high surface area but also facilitate the diffusion and mass transport of reactants. The as-prepared biosensor shows high affinity to acetylthiocholine (ATCl) with a Michaelis–Menten constant value of 0.73mM. Furthermore, based on the inhibition of the enzymatic activity (immobilized AChE) caused by the model compound of carbaryl (one kind of pesticides), it is found that the inhibition activity of carbaryl is proportional to its concentration ranging from 0.001 to 0.05μgmL−1. The developed biosensor shows a detection limit of 0.5ngmL−1 for OPs detection and exhibits good performance such as reproducibility and stability, which makes it possible to provide a new and promising tool for the analysis of enzyme inhibitors.