Aptamers are an excellent choice for the selective detection of small molecules. However, the previously reported aptamer for chloramphenicol suffers from low affinity, probably as a result of steric hindrance due to its bulky nature (80 nucleotides) leading to lower sensitivity in analytical assays. The present work was aimed at improving this binding affinity by truncating the aptamer without compromising its stability and three-dimensional folding. Shorter aptamer sequences were designed by systematically removing bases from each or both ends of the original aptamer. Thermodynamic factors were evaluated computationally to provide insight into the stability and folding patterns of the modified aptamers. Binding affinities were evaluated using bio-layer interferometry. Among the eleven sequences generated, one aptamer was selected based on its low dissociation constant, length, and regression of model fitting with association and dissociation curves. The dissociation constant could be lowered by 86.93% by truncating 30 bases from the 3′ end of the previously reported aptamer. The selected aptamer was used for the detection of chloramphenicol in honey samples, based on a visible color change upon the aggregation of gold nanospheres caused by aptamer desorption. The detection limit could be reduced 32.87 times (1.673 pg mL−1) using the modified length aptamer, indicating its improved affinity as well as its suitability in real-sample analysis for the ultrasensitive detection of chloramphenicol.