Glucosinolates are important secondary metabolites in Brassica vegetables, and the side-chain modifications of aliphatic glucosinolates are regulated by 2-oxoglutarate-dependent dioxygenases (AOPs). However, how the side-chain modifications of aliphatic glucosinolates are regulated in Chinese kale remains unclear. Here, we show that the novel gene BoaAOP-like affects the biosynthesis of aliphatic glucosinolates in Chinese kale. BoaAOP-like was localized in the cytoplasm of Chinese kale. The expression levels of BoaAOP-like increased with plant growth; among different organs, BoaAOP-like was the highest in the roots. BoaAOP-like was introduced into Chinese kale using Agrobacterium-mediated transient overexpression system, of which the expression levels were 2.20- to 49.56-fold higher in transiently overexpressed plants than in wild-type plants; the content of total aliphatic glucosinolates in transiently overexpressed plants was up to 70.26-fold higher than in wild-type plants. The content of each aliphatic glucosinolate was significantly accumulated in BoaAOP-like transiently overexpressed plants, with the gluconapin content increasing the greatest when compared to the wild-type plants. The expression levels of aliphatic glucosinolate-related genes, especially MYB28 and AOP2.1 were significantly up-regulated in transiently overexpressed plants. Overall, our results indicated that BoaAOP-like plays a critical role in regulating the biosynthesis of aliphatic glucosinolate in Chinese kale.