The invention discloses an ecological textile toxic and harmful substance detection and purification column and a preparation method thereof. An outlet screen plate is pressed into the bottom of a column body, gold nanoparticle modified multi-amino-group bonded silica gel (adsorbent) is charged, a layer of isolation screen plates is pressed, then a multi-acidic-group modified polymer crosslinked polymer (adsorbent) is charged, and finally an inlet screen plate is pressed. An extraction column outlet is formed in the bottom of the column body. The column is used for extraction and purification of samples subjected to residue detection of toxic and harmful substances in ecological textiles. The solid-phase extraction column can purify complicated sample matrixes such as cloth, gauze, hemp, silk, leather and wool according to a selective adsorption and selective elution principle, remove azo dyes, amino acids, protein, fat and heavy metal and other impurities, provides a clean sample for GC, HPLC, GC-MS, HPLC-MS and analysis instruments, can prolong the service life of analytical instruments, save costs and ensure the accuracy of poisonous and harmful substance detection.