The invention relates to a preparation method of a temperature-sensitive response polymer array and application of the temperature-sensitive response macromolecule array to pretreatment of samples. Based on a shadow printing technology and an alkynyl and sulfydryl click chemistry technology, a hydroxyl hydrophilic microarray is established on an ITO slide, and then a controllable hydrophilic array of polymer poly-(N-isopropyl acrylamide) (PNIPAM) is generated by surface-initiated atom transfer radical polymerization in the hydrophilic microarray. When the temperature is lower than the critical temperature, due to the hydrophilic interaction of the polymer array, the samples are gathered on the surface of slide; when the temperature is higher than the clinical temperature, a protein/peptide fragment is absorbed by a hydrophobic polymer array due to the hydrophobic interaction, and salt and pollutants coexisting are washed away, so that the pretreatment of on-target self-desalting and efficient enrichment is performed on the samples, and MALDI-MS one-step rapid detection is performed on the target samples. The preparation method is simple in preparation and operation, high in universality, short in analysis time and low in cost, therefore, the preparation method has a certain biomedical application value.