Aeration or oxidation is one of the crucial steps during the plant-derived natural indigo dye production process. However, the lack of scientific information to determine adequate aeration conditions during natural indigo dye production is one of the significant limitations of the process. Therefore, the present study focuses on selecting suitable parameters to assess the optimum oxidation for indigo dye formation. Initially, the effect of direct oxidation parameters, such as dissolved oxygen (DO), oxidation-reduction potential (ORP), and pH on indigo dye formation, were evaluated. However, insignificant ( p > 0.05) co-relation was observed between the direct parameters on indigo dye formation. Alternatively, due to the agglomeration and water insolubility properties of indigo dye, particle size measurement was considered for real-time monitoring of indigo dye formation. It was established that the minimum indigo particle agglomeration size of ≥ 100 μm could be considered as a significant parameter to determine the indigo dye formation. It can act as an indirect indicator of adequate oxidation.