A competitive colorimetric assay has been established to detect chloramphenicol (CAP). It is based on the use of colloidal and electrostatically stabilized aptamer-modified gold nanoparticles (GNPs). The CAP aptamer is modified by a sequence of 5 adenosine groups to anchor it on the surface of GNPs. It can competitively capture two compounds, viz. D-(-)-threo-2-amino-1-(4-nitrophenyl)-1,3-propanediol (CAP-base, with a positive charge) and CAP (which is uncharged). The capture of the positively charged CAP-base triggers the aggregation of modified GNPs in salt-containing solution, and this causes a color change from red to purple. However, in the presence of CAP and CAP-base, the capture of the uncharged CAP weakens this color change by a competing process for capture. Thus, the concentration of CAP is associated with the degree of deaggregation of GNPs and can be quantified by the ratio of absorbances at 620 nm and 520 nm. The assay has a 22 nM limit of detection in acidic solution, and the response is linear in the range of 0.20 to 3.20 μM CAP concentration. This assay was successfully applied to the determination of CAP in spiked environmental water samples. Conceivably, this method has a wide scope in that it may be applied to a wide range of analytes if respective aptamers are available. Graphical abstract Schematic presentation of a competitive non-cross linking deaggregating method for detecting chloramphenicol. The surface charge of polyA-Apt@GNPs and its aggregation degree (purple) are determined by the charge of target. (CAP-base: precursor of CAP; PolyA-Apt@GNPs: 5'-polyA-modified DNA aptamer functionalized gold nanoparticles.).