Acetylcholinesterase (AChE) plays a crucial role in biological neurotransmission. The aberrant expression of AChE is associated with various neurodegenerative diseases. Therefore, it is of great significance to develop a simple and highly sensitive AChE analysis platform. Herein, a simple colorimetric sensor was constructed for sensitive detection of AChE based on the peroxidase-like catalytic activity of Ni/Co layered double hydroxides (Ni/Co LDHs). In this sensor, the fabricated Ni/Co LDHs possess high peroxidase-like activity, enabling rapid catalysis of o-phenylenediamine (OPD) to produce yellow oxOPD in the presence of H2O2. This peroxidase-like activity of Ni/Co LDHs was found to be effectively inhibited by the presence of AChE. It is speculated that the combination of AChE on the outer surface of Ni/Co LDHs through non-covalent interaction may cover the active sites and hinder their adsorption to the substrates, leading to the failure of OPD oxidation. As a result, the yellow color from oxOPD is related to the AChE concentration, enabling the direct AChE assay in an equipment-free manner. In addition, the fabricated Ni/Co LDHs could be modified on a paper surface to obtain a paper-based analytical device for visualized colorimetric detection of AChE. The as-proposed sensor shows high sensitivity to AChE with a detection limit down to 6.6 μU mL-1. Therefore, this naked-eye paper-based sensor is capable of on-site and real-time detection of AChE, and has outstanding application prospects in clinical diagnosis and biomedical fields.