Abstract: The analytical test-system for cocaine, benzoylecgonine and norcocaine was developed in ELISA format using molecularly imprinted polymeric nanoparticles (nanoMIPs) as synthetic recognition elements that were produced using solid-phase synthesis approach. The experimental conditions of the assay were optimised using a Box-Behnken experimental design protocol. The detection of free cocaine and its metabolites was performed using a competitive binding assay in the model samples and in blood plasma. There was no cross-reactivity of the developed assay towards paracetamol and caffeine. The developed assay had a picomolar limit of detection of cocaine (LOD = 4.24 pM), which was almost three orders of magnitude lower than the LOD expected from commercial antibody-based ELISA (3.3 nM), other attractive features of a new assay included a long shelf-life, lower economic cost and a short production time. Therefore, it is possible to state that nanoMIPs have the potential to become the recognition elements of choice for the development of a new generation of test-systems and sensors