Objective A novel facile method for on-site detection of main ingredient (e.g. melatonin, vitamin B₆) in health-care food for promoting sleep using thin layer chromatography (TLC) combined with surface enhanced Raman spectroscopy (SERS) was reported. Methods Firstly, main components were separated with substrate in health-care food by TLC initially, the mobile phase was cyclohexane-ethyl acetate-ethanol (5∶3.5∶2, V/V); detected and located by UV under 254 nm. Then trace substances were qualitatively detected by SERS, the excitation wavelength was 780 nm, and the surface enhancer was aqueous phase silver sol. The effect of matrix on the melatonin and vitamin B₆ was investigated by TLC-SERS in the positive experiment, and the detection limits of melatonin and vitamin B₆ were also investigated. In addition, four commercially available health-care food products to improve sleep were tested, randomly. Results The characteristic peaks of Raman spectra of melatonin and vitamin B₆ obtained by TLC-SERS method were nearly consistent with the reference substance. The matrix of health-care food to add ingredients was no interference on the target substance. The limit of mole tection (LOD) of melatonin and vitamin B₆ were 10 and 5 μg. The TLC-SERS of main components was the same with melatonin or vitamin B₆ substantially. Conclusion This method was accurate, sensitive, fast, and simple. It provides an effective method for the qualitative identification and detection of complex components such as mixed samples.