Linoleic acid as a kind of novel functional oil, has received more and more attention. Sample pretreatment methods for linoleic acid in chlorella powder by GC analysis were studied in this paper. The optimal pretreatment conditions were established by single factor experiments and an orthogonal experiment. The best pretreatment method was acid-catalyzed methylation. The optimum parameters were listed as follows: 2mL of N-hexane and isopropyl alcohol l(1∶1), Reaction time 1hour, 3mL of 2% H2SO4 - methanol solution for methylation, 1mL of ether for methyl linoleate extraction. Under these conditions, the content of linoleic acid was 3.00mg/g.