Pinolenic acid is a polyunsaturated fatty acid present only in Pinus koraiensis Sieb. et Zucc seed oil. It can inhibit and eliminate the adverse effects of other fatty acids on the body. In order to solve the structural instability problem of polyunsaturated fatty acids, pinolenic acid of P. koraiensis seed oil was effectively isolated and purified by the integrated strategy of ethyl esterification followed by urea inclusion for the first time. Under the optimal conditions after the Box-Benhnken Design experimental, ethyl pinolenate with high purity 94.95% could be obtained, and the average content of PNAEE can still reach 86.18%. Then ethyl pinolenate was characterized by Gas chromatography-mass spectrometry, Fourier transform infrared, and Nuclear magnetic resonance spectra, results showed that ethyl pinolenate was successfully prepared. In addition, the hypolipidemic activity of ethyl pinolenate had been tested in vivo and showed that ethyl pinolenate had obvious hypolipidemic activity. The new strategy for high purity ethyl pinolenate production from P. koraiensis seed oil possesses great potential in food healthy field in the future.