Conjugated linolenic acid (CLnA) and conjugated linoleic acid (CLA) are positional geometric isomers with three and two double bonds, respectively. In this study, perilla seed oil containing 60% α-linolenic acid (C18:3) and 30% linoleic acid (C18:2) was used as a reaction substrate. After the perilla seed oil was hydrolyzed, conjugated fatty acids were synthesized using different reaction parameters, such as reaction time and concentration of sodium hydroxide. As a result, CLnA, CLA, and other newly synthesized conjugated isomers were present at levels of 14.5%, 14%, and 42.2%, respectively, when the reaction was performed with 20% NaOH, at 180°C, and for 1 hr. The results of GC-MS and fourier transform infrared spectroscopy (FT-IR) showed that CLnA isomer of cis-9, trans-11, and trans-13 octadecatrienoate, CLA isomer of cis-9, trans-11, and trans-10, cis-12 octadecadienoate, and other conjugated isomers were produced. Using urea, α-linolenic acid could be concentrated from perilla seed oil hydrolysate. After concentration by urea, the concentration of α-linolenic acid reached about 70%. After alkaline-isomerization was performed on the urea fraction containing 70% α-linolenic acid, the content of CLnA increased up to 16.6%.