In this study, functionalized hydrogel bioadsorbents were produced from gum tragacanth (GT) carbohydrate and quaternary ammonium salt (TMSQA) as a crosslinker. The prepared bioadsorbents were used for the removal of NO3− ions from water through the electrostatic and ion exchange mechanism and antibacterial activity. The effect of quaternary ammonium content on the adsorption capacity was studied. The bioadsorbents were characterized by using FE-SEM, energy dispersive X-Ray (EDX), FT-IR, and TGA techniques. The equilibrium time and the most effective pH value for maximum NO3− removal (20 mg g−1) were 21 min and 7, respectively. A series of isotherms and kinetics models were undertaken and the obtained data were fitted well to the Langmuir isotherm and pseudo-second-order rate kinetic. The thermodynamic study confirmed the suitability of NO3− removal by the as-prepared bioadsorbent at room temperature, and also the negative value of ΔGº = −89.1 kJ mol−1 demonstrating the spontaneous nature of adsorption.