On considering the critical issues in attaining stringent water quality standards and not creating any environmental impacts, we focused for the first time the economically feasible, emerging technology known as Self-assembly flocculating (Saf process). In which, the study investigated the applicability of bioflocculant (a biopolymer-self-assembly in nature) act as a surrogates on relying the removal of broad spectrum of substances under optimized conditions (Dosage: 90 mg/L; pH: 7; CaCl2). On using different techniques, the results have proved in removing the organic matter such as pharmaceuticals (Gentamycin, Cholecalciferol, Fluvoxamine, 3-OH Desogestrel, and Pheniramine), endocrine disturbing compounds [Phthalic acid, Benzene, 1, 2, 4 -Trimethoxy-5-(1-Propenyl)-, Benzene, 1, 2-Dimethoxy-4-(2-Propenyl)-, 1, 2-Benzenedicarboxylic Acid, 3-Cyclohexen-1-ol], fluorescent components (Polysaccharide like material), and others. The toxicological assessment of self-assembly bioflocculant implemented on zebra fish were statistically correlated [r = 0.95, p < 0.01 and 0.05 for P1WW; r = 0.91, p < 0.01 and 0.05 for P2WW] and [r = 0.7 5, p < 0.05 for P1WT; r = 0.095, p < 0.01 and 0.05 for P2WT]. This integrated approach supplemented further information of zeta potential (−16 mV in P1WW and −14.6 mV in P2WW decreased to −1.05 mV and −1.56 mV) with particle size distribution to explain via Saf process. In this research, the new insight has established non-toxic, self-assembly, biodegradable, bioflocculant for effective bioremediation.