This study aimed to assess the effects of different linolenic acid (LNA, 18:3n-3) and linoleic acid (LA, 18:2n-6) ratios in diet on growth performance, fatty acid composition in muscle and liver and non-specific immunity in serum, liver and intestines of juvenile Manchurian trout (Brachymystax lenok). With casein, soybean meal and corn protein meal as main protein sources, a vegetable oil-based (VO-based) diet with iso-nitrogen (40%) and iso-lipid (18.5%) was prepared by adding different proportions of linseed oil, corn oil and palm oil. The LNA/LA ratio in the VO-based diets was 0.02, 0.55, 1.06, 1.61 and 2.17 (constant composition of LNA and LA). The control group (CON) used fish oil (FO) as the sole lipid source. Healthy juvenile Manchurian trout (body weight: 7.00 ± 0.07 g) were fed in a recirculating aquaculture system for 9 weeks. The results showed that VO-based diets did not affect the growth of Manchurian trout, but decreased DHA and EPA levels in the dorsal muscle of Manchurian trout. Levels of LA, arachidonic acid (ARA) and n-6 polyunsaturated fatty acids (PUFA) of liver in VO-based diets were significantly higher than those in CON diet and decreased with an increasing LNA/LA ratio in the diet. The composition of LNA, DHA, EPA and n-3 PUFA of dorsal muscle and liver in VO-based diets increased with increasing LNA/LA ratio. In the liver and proximal intestine (PI), lysozyme (LZM) activity increased with the increasing LNA/LA ratio in the VO-based diet. However, it decreased in the mid-intestine (MI) and distal intestine (DI). Acid phosphatase (ACP) activity in liver was significantly high when the LNA/LA ratio in the diet was 1.06 (p < .05). Alkaline phosphatase (AKP) activity in the MI increased with the increasing dietary LNA/LA ratio. The LNA/LA ratio in the diet did not affect the growth of Manchurian trout juveniles, and a diet with an LNA/LA ratio between 0.55 and 1.06 was beneficial for non-specific immunity in Manchurian trout.