The sludge yield minimization from advanced biological treatment for industrial wastewater could be considered a poorly explored area, therefore, seeks serious attention of the scientific community. Up to best of the knowledge, the extracellular polymeric substances (EPS) profile underlying an upgraded activated sludge treatment (as MANODOX system) for real tannery wastewater has not been addressed in a desired manner. This study covers the elucidation of EPS degradation mechanism and floc morphology underlying MANODOX system for the treatment of real tannery influent. For this purpose, a modified heat extraction method was followed for the estimation of EPS fractions like protein (PN), polysaccharides (PS) and humic contents from the sludge. For the present investigation, the variation in floc characteristics including PN/PS ratio, sludge hydrophobicity, sludge volume index, and facultative microbiota at corresponding change in hydrodynamic sludge retention time (SRT) of 08–40 days was emphasized. The strict maintenance of adapted operational strategies including favoring range of SRT (24 days) for MANODOX implementation succeeded an outstanding in-situ sludge yield minimization lowered up to 0.39 gMLSS/gTCOD that attributed to three times lowered accumulation of PN and PS, comparably lower PN/PS ratio, higher salinity of the mixed liquid, weakened cell-to-cell attachment compared with a parallel run identical aerobic treatment. Here, the reason for improved hydrophobicity and corresponding decline in floc aggregation was attributed to change in sludge PN/PS ratio, carbon to nitrogen ratio of feed influent. The observations confirmed that the sludge yield minimization from MANODOX like systems could be effectively controlled by maintaining aforementioned operational tactics.