Thin-layer chromatography (TLC) is widely used in various branches of chemical science to separate components in complex mixtures because of its simplicity. In most cases, analyte spots are visually detected by fluorescence, and the retention factor (Rf) is determined from the distance traveled by the analyte. Further characterizations are often necessary to identify separated chemicals because molecular information other than Rf is not available. Surface-enhanced Raman scattering (SERS) has been coupled with TLC to complement molecular information. In previously reported TLC-SERS, metal nanoparticle suspension was dropped onto analyte spots to obtain SERS spectra. This approach is simple and efficient for SERS measurements on the TLC plate but has limited sensitivity for several reasons, such as the low solubility of analytes in the dropped solution, difficult control of nanoparticle aggregation, and interference from the stationary phase. We recently showed that freezing enhances SERS sensitivity by a factor of ∼103. Freezing simultaneously concentrates analytes and silver nanoparticles (AgNPs) in a freeze concentrated solution, where aggregation of AgNPs is facilitated, allowing sensitive freeze SERS (FSERS) measurements. Here, we discuss FSERS measurements on TLC plates to demonstrate the superiority of this combination, i.e. TLC-FSERS. Freezing enhances SERS sensitivity by freeze concentration and facilitated aggregation of AgNPs and, in addition, eliminates interference from the stationary phase. Under the optimized condition, TLC-FSERS enables the on-site detection of pesticides at the nM level. The use of the SERS signal from adenine added as the internal standard allows us to quantify pesticides. Applications to a commercial green tea beverage are also demonstrated.