As one of Maillard’s hazards, acrylamide (AAm) can induce neurotoxicity, mutagenicity, and genotoxicity, and it’s urgent to construct a detection method for sensitive and specific monitoring of AAm. Herein, an aptamer-gated colorimetric sensor was developed to detect AAm in food. UiO-66-NH2 was selected to combine acetylcholinesterase (AChE) and the aptamer of AAm to construct the aptamer-gated probe UAzyme-Apt. The addition of AAm would detach Apt and expose the active sites of UAzyme, resulting in the transformation of acetylthiocholine (ATCh) into thiocholine (TCh) that could inhibit the oxidase mimic activity of MIL-53 (Fe/Mn). Consequently, the concentration of AAm was inversely proportional to the absorbance at 652 nm, thus providing a qualitative and quantitative detection of AAm. The aptamer-gated probe can recognize AAm to improve specificity and stability and regulate the enzyme activity of UAzyme. This aptasensor enhances the detection methods of AAm and broadens the scope of applications for AChE inhibition detection, and targets are not limited to the substrate or organophosphorus pesticides. The linear range and limit of detection were 0.05–100 μM (R2 = 0.992) and 9.94 nM, and spiked analysis revealed the efficiency and robustness of the developed assembly. The colorimetric aptasensor can specifically detect AAm at low concentrations, and will be a promising tool to ensure food safety in the coming future.