Long chain omega-3 polyunsaturated fatty acids (LC n-3 PUFA) are beneficial for health. To date there is no specific food frequency questionnaire (FFQ) to assess LC n-3 PUFA intakes. The objective of this study is to validate our newly developed FFQ by comparison with LC n-3 PUFA content of both red blood cells (RBC) and plasma, expressed as a percentage of total fatty acids. Fifty-three healthy male and female subjects were recruited from Wollongong, Australia. Average LC n-3 PUFA intakes (mg/day) were estimated using the new FFQ. RBC and plasma fatty acids were assessed using gas chromatography. Spearman correlation co-efficients assessed the linear relationship between FFQ intakes and both RBC and plasma fatty acids. ) The results show that there were significant Spearman’s correlation co-efficients between the FFQ intakes and RBC (and plasma) fatty acids for total LC n-3 PUFAs, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) (0.50 (0.54), 0.39 (0.54) and 0.40 (0.48) respectively) but not for docosapentaenoic acid (DPA). The FFQ was also an effective ranking tool. In summary, the FFQ is a valid method based on erythrocyte and plasma fatty acids as biochemical markers. In conclusion, the new FFQ is a valid method that can be used to estimate the LC n-3 PUFA intake of adults.