Gas chromatography-mass spectrometry (GC-MS) was used to analyze fatty acid profile in metabolites and cells of five strains of Bifidobacterium. The results showed that the fatty acid content of different Bifidobacterium was significantly different among the metabolites, and there were some differences among the cells. Differences of fatty acid were also found among the same Bifidobacteriium strains derived from different sources. Methyl palmitate (C_16:0), methyl lignocerate (C_24:0), and methyl stearate (C_18:0) were the main fatty acids in metabolites and cells of the five strains. Among them, the C_16:0 was significantly different (P＜0.05), and the content ratio of C_16:0 to C_18:0 was between 2.607 and 3.120; the content ratio of acetic acid to lactic acid was between 2.085 and 2.295. Compared with the metabolites, methyl erucate (C_22:1n-9), methyl nervonate (C_24:1n-14), methyl 5, 8, 11, 14, 17-eicosapentaenoate (C_20:5n-3) were not detected in the cells of the five strains.