A method was developed and validated for the analysis of docosahexenoic acid (DHA) in human blood by heterocyclic derivatization-gas chromatography coupled with triple quadrupole mass spectrometry (GC-MS/MS). 2-Amino-2-methyl-1-propanol (AMP) was used as the reaction reagent of DHA heterocyclic derivatization and the most optimal reaction conditions of this reaction were optimized. Multiple reaction monitoring and internal standard calibration curve were applied to detect DHA by GC-MS/MS. The linear range for the determination of DHA was 0.07 - 10 microg/mL (r(2) = 0.9991). The limit of detection (S/N = 2.8) was 0.02 microg/mL and the limit of quantification (S/N = 10) was 0.07 microg/mL. The average recoveries of DHA at three spiked levels of 0.5, 1.5 and 2.5 microg ranged from 94.40% to 103.13% and the relative standard deviations (RSD) were in the range of 1.51% - 3.16%. The method was simple, accurate, reliable and small amount of sample was required. It was suitable for detecting the contents of DHA in human blood.