Abstract: Aminopiperazine functionalized dansyl chloride (DNS-CL) fluorescent derivatization reagent (DNS-Pi-NH₂) was self-made, and used for derivatizing typical fatty acids containing 10-20 carbon atoms (dodecanoic acid, tetradecanoic acid, cis-9,12-octadecenoic acid, cis-7,10,13-hexadecenoic acid, hexadecanoic acid, cis-9-octadecenoic acid, octadecane acid, eicosanoic acid) in Chlorella, and the 8 fatty acids were determined by high performance liquid chromatography (HPLC). Chlorella fatty acid methyl ester sample was saponified with ethanol solution containing potassium hydroxide at 79℃ for 60 min, and hydrochloric acid solution was added dropwise until the phenolphthalein indicator changed from red to colorless. The ethanol was removed by evaporation, and the solid obtained was washed with water, centrifuged, vibrated and filtered to prepare Chlorella fatty acid samples. DNS-Pi-NH₂ was prepared from DNS-CL and N-(2-aminoethyl) piperazine, and its structure was verified by infrared spectroscopy, nuclear magnetic resonance carbon spectroscopy and mass spectrometry. Dipropyldisulfide, acetonitrile solutions of DNS-Pi-NH₂ and triphenylphosphorus were mixed with 1.0 mg Chlorella fatty acid sample, and the mixture was shaken at room temperature for 10 min to derivatize. The obtained solution was diluted with 90% (volume fraction, the same below) acetonitrile solution, and derivatives of fatty acids in which was determined by HPLC. Eclipse XDB C₈ column was used as stationary phase, 90% acetonitrile solution was used as mobile phase for isocratic elution, and fluorescence detector was used for detection. As found by the results, the derivatives of 8 fatty acids could be completely separated within 30 min. The concentration of each fatty acid was linearly related with its corresponding peak area in the range of 2.0×10^-10-2.0×10^-4mol·L^-1, with detection limits (3s/k) of 21-65 pmol·L^-1. Test for the spiked recovery was made on microalgae culture medium, giving recoveries in the range of 95.3%-102%, and RSDs (n=5) of the determined values ranged from 1.7% to 2.6%. The proposed method was applied to the analysis of Chlorella fatty acid methyl ester sample, and hexadecanoic acid, cis-9,12-octadecanoic acid, cis-7,10,13-hexadecenoic acid and cis-9-octadecanoic acid were detected, with detection amounts of 3.18, 0.64, 0.83, 2.57 mg·g^-1, respectively.