This study investigates the effect of graded substitution of dietary fish oil (FO) with canola oil (CO) on fatty acid composition and expression of Δ-6 desaturase and elongase 2 genes in the muscle of Jade Tiger hybrid abalone. The control diet contained 1.5% FO supplement (0% CO). Four other diets contained FO/CO in ratios of 3:1 (CO 25%), 1:1 (CO 50%), 1:3 (CO 75%) and 100% CO. The result demonstrated that abalone fed the diets supplemented with 25% and 50% CO showed similarly high levels of total omega-3 polyunsaturated fatty acid (n-3 PUFA), eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3) (P < 0.05) as the 0% CO group. The highest levels of total PUFA and total monounsaturated fatty acids were found in the 100% CO group (P < 0.05). The results also showed that Δ-6 desaturase and elongase 2 gene expression was increased in a graded manner by increasing dietary CO (P < 0.05). It was concluded that replacement of FO by CO at the levels of 25–50% can improve the contents of health-benefiting EPA and DHA in abalone.