This study introduces CRISPR/Cas-based aptasensor for the highly sensitive and specific detection of the antibiotic, ampicillin. Ampicillin (AMPI) is a commonly used antibiotic for treating pathogenic bacteria and is additionally added to livestock feed in agriculture. This study can enable early detection of antibiotic residues, prevent their accumulation in the environment, and ensure compliance with food safety regulations. Herein, the aptasensor was developed with the CRISPR/Cas system by utilizing three different ampicillin-specific aptamers, each conjugated with a biotin at the 5′-end. The ssDNA activator was bound to the aptamers through complementary base pairings. The attraction of the aptamers to the ampicillin target released the bound ssDNA, causing the activation of the CRISPR/Cas system. The DNA reporter probe, labelled with Cy3 and a quencher, turns on the fluorescence signal when cleaved by the activated Cas12a through trans-cleavage measured using a fluorescence spectrophotometer at 590 nm. The fluorescence signal was linearly proportional to the ampicillin target concentration with a 0.01 nM limit of detection and a read-out time of 30 min. This aptasensor showed high sensitivity towards ampicillin even in the presence of other antibiotics. The method was also successfully implemented for ampicillin detection in spiked food samples.