This study was conducted to investigate the effects of dietary incorporation of soybean meal for fish meal replacement and supplementation of betaine as an attractant on growth performance and fatty acid profiles of rainbow trout (Oncorhynchus mykiss). Juvenile trout were fed practical diets, with increasing soybean levels and betaine supplementations. The experimental diets consisted of a control diet with fish meal as the sole protein source (control) and no attractant, 25% soybean‐1% betaine diet (SBM25‐B1), 50% soybean‐2% betaine diet (SBM50‐B2), and 50% soybean‐0% betaine diet (SBM50‐B0). Average body weight was 12.69 ± 0.16 g at the beginning of experiments. Following 54 days feeding programme with experimental diets, average body weights reached 47.45 ± 1.22 g, 58.11 ± 1.77 g, 56.34 ± 1.87 g and 53.76 ± 1.74 g in the control, SBM25‐B1, SBM50‐B2 and SBM50‐B0 groups respectively. As compared with control treatment, significant differences were observed in weight gain, specific growth rate and feed intake of 1% betaine treatment at 25% soybean‐meal‐incorporated diet (p < .05), but no differences were observed in feed conversation ratio and survival rates (p > .05). Compared with the control treatment, betaine‐supplemented groups had significantly higher total saturated fatty acid contents (p < .05). On the other hand, the control treatment showed a significantly higher level of monounsaturated fatty acid than the betaine‐supplemented groups (p < .05). Significant differences were observed in fatty acid profile of 1% betaine‐supplemented group (p < .05) compared with the control. Present findings revealed that 1% betaine supplementation with dietary incorporation of soybean meal at 25% level positively influenced growth performance, feed utilization and fatty acid profiles of rainbow trout juveniles.