Abstract: HPLC with sample treatment by solid phase micro-extraction (SPME) was applied to the determination of malachite green in water samples. To attain a higher efficiency in SPME, the 85 μm PA coating was applied to the micro-extractor, and the extraction was carried out for 15 min. The Shim-pack VP-ODS C18 column was used as the stationary phase, and a mixture of 50 mmol·L-1 ammonium acetate solution of pH 4.5 and acetonitrile (20+80) was used as the mobile phase for elution. Detection at 595 nm was adopted. Linearity range of malachite green was kept in the range of 0.05-2 mg·L-1, with detection limit (3S/N) of 0.003 mg·L-1. A sample of fish pond water was analyzed by this method, and test for recovery was made on this sample as matrix by standard addition method giving results in the range of 98.1%-109.6%.