The UV−VIS absorption spectroscopy technique was used to study the formation of a new complex of charge transfer (CT) between bioactive organic molecules as (Nystatin) containing both a π-electrons from a conjugated system and lone-pair of electrons (amine) with Tetrachloro-1,4 benzoquinone (TCBQ) as a π-acceptor in which the transferred electron goes into its vacant anti-bonding molecular orbitals. The Tyrian purple-colored complex formed was quantitatively measured at 544 nm. This complex shows obeying Beer's law within the concentration range of (10-90) μg.ml-1The stoichiometry of the formed complex between the (Nys.) and (TCBQ) was found 1:2 as evaluated by continuous variation (Job's method) and mole ratio method The value of molar absorptivity was calculated at 7038.2840 L.mol-1.cm-1, while Sandell’s sensitivity value was estimated to be 0.01315 μg.cm-2, while LOD and LOQ were found to be 0.5661and 1.71558 μg.ml-1, respectively. The Charge-Transfer complex association constant (KCT) value was evaluated using Benesi-Hildebrand equation and was found to be 3.00E+03 L.mol-1. This procedure was successfully involved in the analysis of pharmaceutical formulations.