The cyclopoid copepod Apocyclops royi (Lindberg 1940) is one of two dominant mesozooplankton species in brackish Taiwanese aquaculture ponds. Especially periodical low n-3 polyunsaturated fatty acids (PUFA) content of seston could potentially be a limiting factor for the zooplankton diversity. A. royi’s potential ability to biosynthesize n-3 PUFA was investigated through a short-term feeding experiment on four species of microalgae. Furthermore, we analyze the expression of genes encoding putative fatty acid elongase (ELO) and desaturase (FAD) enzymes in A. royi on long-term diets of the PUFA poor D. tertiolecta and the PUFA rich I. galbana. The copepods exhibited high contents of docosahexaenoic acid (DHA, C22:6n-3) (> 20% of total fatty acid) even when DHA-starved for two generations, and no significant differences were found in absolute DHA content between treatments. Transcripts correlating to enzymes in the n-3 PUFA biosynthetic pathway were identified: Elovl4, Elovl5, Fad Δ5 and Fad Δ6. Gene expression analysis revealed a significantly higher expression of two desaturases similar to Fad Δ6, in copepods fed PUFA-lacking algae compared to copepods fed algae with high PUFA content. These findings suggest a highly active n-3 PUFA biosynthesis and capability of DHA production of A. royi when fed low-PUFA diets.