Honesty (Lunaria annua L.) is an oilseed crop that needs to be developed urgently, and its seeds contain a large amount of nervonic acid that can prevent cerebral neurasthenia. Currently, there are no reports on the characteristics of fatty acid accumulation and metabolism during the fruit development process of Lunaria. In order to explore the changes in the types and contents of fatty acids during the development process of the fruit of Lunaria, as well as the synthesis characteristics of very-long-chain fatty acids, especially nervonic acids, this study used acid-base combination method and GC-MS to compare and analyze the fatty acid components and content of plants containing nervonic acids: Lunaria, Arabidopsis, Borage, and Malania fruit. The results showed that a total of 10 types of fatty acids were involved in fatty acid metabolism during the development process of the Lunaria fruit, seven in seeds and eight in pericarps, most of them are long chain and very-long-chain unsaturated fatty acids. The fatty acids in the seeds were mainly composed of palmitic acid, stearic acid, oleic acid, linoleic acid, linolenic acid, erucic acid and nervonic acid; the content of palmitic acid, stearic acid, linoleic acid and linolenic acid gradually decreased with seed development; oleic acid, erucic acid and nervonic acid gradually increased with seed development and accumulated in large quantities in the first 25-35 days after anthesis; the pericarp was free of erucic acid and nervonic acid, but specializes in behenic acid, lignoceric acid and cerotic acid. Lunaria, Borage and Malania seeds all contain erucic acid and nervonic acid. Woody plant Malania seed has the highest content, followed by Lunaria, and Borage has the lowest content. The content of nervonic acid in the seeds of the three plants is significantly positively correlated with the content of erucic acid. The results of this study elucidate the characteristics of fatty acid metabolism and accumulation of nervonic acid during the fruit development process of Lunaria, providing new ideas for fully utilizing the germplasm resources of Lunaria and expanding the pathways for obtaining nervonic acids.