Humic acid, a natural organic matter, was separated with a polyamide (PA) composite membrane with a molecular weight cutoff (MWCO) of 500 g/mol. The PA composite membrane was prepared by the interfacial polymerization of piperazine and trimesoyl chloride on a polysulfone support with an MWCO of about 30,000 g/mol. The separation conditions through the membrane were varied, and factors affecting the permeation performance of the membranes, such as the concentration, pH, and storage time of the humic acid solutions, were studied. The surface chemistry of the membrane changed dynamically as a function of the operating time during the permeation tests, and the size and zeta potential of the colloid of humic acid solutions under different conditions were characterized with a zeta potentiometer.