The thin-layer chromatography (TLC) procedures of metal cations, modified with amines and cationic surfactant micelles, followed by the sorption-catalytic detection of cobalt(II) and copper(II) directly into the thin layer were presented. It has been shown that the addition of hexamethylenediamine to the mobile phase acetone—3 M HCl and the impregnation of TLC plates with cetyltrimethylammonium bromide micelles and triethylamine followed by separation in 5% NaCl (pH 2.3) improves the separation efficiency of divalent metal cations mixture. A new highly sensitive detection of copper(II) and cobalt(II) after modified TLC with sorption-catalytic method was proposed. The TLC modifiers enhance the catalytic effect of copper(II) and cobalt(II) in the indicator reactions and detection selectivity. The detection limit was 0.1 ppb for copper(II) and 0.02 ppt for cobalt(II). It should be noted that cobalt(II) does not interfere with the determination of copper( II) in a 200-fold excess. The possibility of using the developed procedures in the analysis of natural water and food was shown.