Abstract In Brazil, about 25-30% of the population has some degree of intolerance to lactose, a disorder associated with the inability of the body to digest lactose due to a disability or absence of the enzyme lactase. The goal of this study was to evaluate the performance of adsorption of lactose from fresh milk using a fixed bed column of molecularly imprinted polymer (MIP). The polymeric material was characterized using Scanning electron microscopy (SEM) analysis, thermal analysis (e.g., differential scanning calorimetric (DSC) and thermogravimetric analysis (TGA), Fourier Transform Infrared Spectroscopy (FTIR), and the method of Braunauer, Emmet and Teller (BET). The adsorption column dynamics and performance were studied by the breakthrough curves using a 24-1 fractional factorial design. The chemical and structural characterization of the pure matrix and imprinted polymers confirmed the molecularly imprinted polymer (MIP) imprinted with lactose. The highest capacity was 62.21 mgg-1, obtained at 307.1 K and a flow rate of 12.5 mL.min-1, with central point conditions, 320.1 K and 9 mL.min-1, with an average value of 50.9 mg.g-1. The results indicate that the molecularly imprinted polymer is efficient.