The invention belongs to the technical field of plant gene engineering, and in particular, relates to an expression vector for comprehensive down-regulation of cotton lycopene cyclase (Lyc) genes and an application thereof. The technical scheme comprises that coding sequences selected from 5 pairs of cotton Lyc genes respectively construct an RNAi element in series; the element is transcribed in cotton by a transgenic method, and ultimately, the purposes of down-regulating the 5 pairs of Lyc genes and regulating synthesis of carotenoids in the cotton simultaneously are achieved. The RNAi element and the vector can be used for regulating the content and type of the carotenoids in the cotton.