A simple, sensitive and highly selective detection method for nalidixic acid and flumequine has been developed. A 1-μL aliquot of sample solution was spotted onto the thin-layer plate with the aid of a disposable micropipette, and after development, the plate was sprayed first with sodium borohydride and then hydrogen peroxide successively. The spots were visualized under longwave UV light. The detection limits were 30 pmol for nalidixic acid and 60 pmol for flumequine.