Abstract In this study, the adsorption and electrokinetic properties of biomolecules on kaolinite were studied as a function of incubation time, temperature, and concentrations of biomolecules, and pH of the solution. Catalase adsorption has caused a change in the zeta potential of the solid particles for kaolinite at approximately pH 5.8. The amount of biomolecule adsorbed on kaolinite increased by increasing temperature (from 15 to 45°C). A full factorial experimental design was utilized to assess the effect of two factors (pH and temperature) on the adsorption of biomolecules. The main effects and 2-way interactions were found to be statistically significant since the P-value, defined as the smallest level of significance leading to rejection of the null hypothesis was <0.05. FTIR spectra results of biomolecules showed strong peaks at v=1620–1680cm−1 and v=1480–1580cm−1. Rate constants of first-order kinetic equation for the adsorption mechanisms of casein and catalase molecules on kaolinite were found as 9.8×10−3 and 4.1×10−3min−1, respectively. Freundlich isotherms for biomolecules adsorption on kaolinite appear to the experimental data better than the Langmuir isotherm. Km and Rmax values of the adsorbed enzyme and pure enzyme were calculated as 29.22mM, 3.33mMmin−1 and 172.09mM, 10.10mMmin−1, respectively. Thermodynamic parameters such as Arrhenius activation energy (Ea), ΔG∗, ΔH∗, and ΔS∗ were calculated as 29.16, 215.2, 35.02kJmol−1 and −611.5Jmol−1K−1 for casein adsorption and 33.12, 175.6, 38.55, and −595.5Jmol−1K−1 for catalase adsorption, respectively.