Cyanide (CN) is a powerful poison and rapidly toxic agent. Because of its wide availability and high toxicity, quantification of CN in blood and urine is frequently required in clinical and forensic practice. We present a sensitive and less time consuming method based on solid-supported liquid-liquid extraction (SLE) and gas chromatography-mass spectrometry (GC-MS) with two-step derivatization for determination of CN in plasma and urine. Buffer solution, 1,3,5-tribromobenzene (internal standard) and benzaidehyde were added to sample to complete the first-step derivatization. Then the analytes were poured onto the column of diatomaceous earth, eluted with n-hexane containing 0.4% of heptafluorobutyryl chloride (HFB-Cl) to complete the second-step derivatization forming the final analyte, heptafluoro-butyric acid-alpha-cyanobenzyl ester. This method was linear (r(2)=0.9988, 0.9993), reproducible (intra-day RSD=4.37-7.24%, 3.19-5.74%; inter-day RSD=5.13-7.63%, 4.31-6.69%), accurate (recoveries=90.58-115.56%, 93.01-114.6%) and sensitive (LOD=0.04, 0.01microg/mL) for plasma and urine, respectively. The total time was about 25min. This method was successfully applied to the analysis of blood sample and urine sample collected from a victim who died as a result of ingestion of potassium cyanide.