Facile and effective coupling of SERS sensor to chromatographic tools has been an attractive goal in the field of food analytical chemistry for years. Platformed by HPTLC, a separable SERS sensor was developed in this study and its analytical power was exemplarily evaluated by screening trace malachite green in fish. To enable enough detectability and reproducibility of SERS measurements, a flexible substrate based on the hybrid of AgNP and glass fiber paper was easily fabricated. Meanwhile, the crude extract of fish muscle was separated on the aluminum foil backed HPTLC plate, resulting in spiral isolation of the analyte from co-extracted matrix. Guided by the visible standard in parallel, bands of interest were thereafter transferred to the eluent by a handy puncher. Jointly with the as-prepared flexible substrate, the band eluent was measured by Raman spectroscopy, responding sensitive and characteristic fingerprint signals. Meanwhile, performances of the established SERS sensor were also examined, showing adequate detectability (LOD < 10 μg/kg), precision (RSD%<11.8%) and linearity (R2 = 0.9975 from 0.05 to 10 ng/band) for screening purpose. Apart from that, the analysis robustness for practical application was further validated with fish sample, giving spike-recovery rates from 78.8 to 87.3%. This work demonstrated that HPTLC platformed SERS sensor can reach an ideal balance between efficiency, throughput and reliability, and therefore might be a promising tool especially suitable for screening trace contaminants in sophisticated mixtures.