The antiscalant for the reverse osmosis(RO) system can inhibit the crystallization and precipitation of Ca2+ and Mg2+, resulting in a high dosage of hydroxide and carbonate, a low removal efficiency, and profoundly negative impacts on the evaporation crystallization of salt. It is crucial to explore the industrial antiscalant ingredient and anti-scaling mechanism in RO system, contributing to the in-depth hardness removal and zero discharge of wastewater. In this study, we have determined the water quality of RO concentrate and fractionated it by ultrafiltration. FTIR, UV-Vis, and EEM spectra results indicated that the dissolved organic matter in RO concentrate was mainly composed of microbial metabolites and humic acid substances with molecular weight <3 kDa, containing carboxyl, alcohol/phenol hydroxyl, and unsaturated hydrocarbon structure. According to 13C and 31P NMR spectra, chemical shift characteristics, including methyl carbon, quaternary carbon, and C-PO3H2 structures, have confirmed the main component of antiscalant is hydroxyethylidene-1,1-diphosphonic acid (HEDP). After removing 88.55% HEDP and 38.86% COD by adsorption, the amount of added carbonate for the complete precipitation of Ca2+ was reduced from 2845.8 mg?L-1 to 826.8 mg?L-1, indicating that HEDP dominantly hindered the Ca2+ crystallization. Even if it is prepared with HEDP again, the amount of carbonate was only increased to 1626.3 mg?L-1, demonstrating that the removed organic matter by adsorption has dispersion effects to inhibit the crystallization and aggregation of Ca2+. The above results have revealed the synergistic anti-scaling mechanism between HEDP and the dissolved organic matter to provide a significant theoretical and regulatory strategy for the in-depth hardness removal in RO concentrate.