A novel spectroscopic probe based on a barbituric acid-starch (ST-BA) derivative is synthesized and used to assess the alpha-amylase activity. This derivative and its precursors are characterized by UV-Visible, fluorescence, FTIR, and 1H NMR spectroscopy. Luminescent ST-BA core contains a double bond conjugated to pyrimidine ring carbonyl groups, yielding a notable UV-Visible absorption and fluorescence emission. Enzymatic ST-BA cleavage reveals a kinetics slower than for pristine starch, inducing new supramolecular structures with an enhanced fluorescence quantum yield. Force field minimization also supports the contribution of intra and intermolecular interactions to ST-BA oligomer rigidification, a singularity in agreement with aggregation-induced emission. However, the decrease of UV-Visible signal reveals the chromophore competition for the photon after oligomer self-assembly. The linear spectroscopic responses support to ST-BA as a successful enzymatic activity sensor. Although these findings emphasize the ST-BA potential for qualitative enzyme analysis, further experimental condition studies are essential for accurate quantification.