The purpose of this study was to evaluate the effect of varying dietary levels of highly unsaturated fatty acids (HUFAs) in live prey (Artemia nauplii and a calanoid copepod, Schmackeria dubia) on the growth performance, survival, and fatty acid composition of the lined seahorse, Hippocampus erectus, juveniles. Artemia nauplii were enriched with a commercial product (SS™ 50DE-microcapsule as HUFA source, 2/3 DHA, 1/3 EPA. Shengsuo Fishery Feed Research Center of Shandong Province, Qingdao, China) at four concentrations of 0.0, 14.0, 28.0, and 56.0. Newly hatched juveniles were cultured for 35 days. The content of docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), and n-3 HUFAs in the Artemia nauplii was positively related to the enrichment concentration. At the end of the trials, growth performance of the juveniles was positively related to the enrichment concentration as well. However, the juveniles fed prey enriched with the highest concentration of enrichment (56.0 μL/L) had the significantly lower (P < 0.05) survival rate. The juveniles fed the copepod had the best growth performance and the highest survival rate, suggesting that the copepod, S. dubia, is suitable for feeding the seahorse juveniles. The comparisons between the growth, survival, and fatty acid profiles of the juveniles fed Artemia and copepods indicate that the seahorse juveniles require dietary levels of DHA beyond those achieved by enriching prey with the HUFA enrichment. Surplus EPA resulted from an imbalance between DHA and EPA in the enriched Artemia nauplii probably caused an adverse effect on the seahorse juveniles. This study suggests that DHA and EPA requirement of the lined seahorse juveniles is roughly 32% of total fatty acid, and the optimal DHA/EPA ratio for the species is circa 4:1. To avoid an adverse effect resulting from excessive EPA, maximum proportion of EPA in enriched Artemia nauplii should not exceed 13% of total fatty acid, and a recommended minimum DHA/EPA ratio in the enriched Artemia nauplii is 1.46. Arachidonic acid (20:4n-6) might not be an essential fatty acid for the seahorse juveniles.