The invention belongs to the technical field of crop molecular breeding, and in particular relates to a method for interspecific hybrid genome in-situ hybridization of crambe cordifolia. The method comprises the following steps: preparing a probe marker, preparing a slide, performing in-situ hybridization, performing signal detection and the like. In the slide preparation process, a chromosome slide which is good in morphology and good in dispersity in the meiosis period of crambe cordifolia can be obtained under appropriate enzymolysis conditions by using an acetic acid extension flame drying method. When probes and chromosomes are modified, appropriate temperature and time can be selected, appropriate blocking DNA and a probe concentration ratio can be confirmed, fluorescent in-situ hybridization images with clear signals can be obtained, and different interspecific chromosomes of hybrid crambe cordifolia species can be clearly identified, so that genome in-situ hybridization can be well applied to cytological detection of crambe cordifolia species with characteristics of a large number of chromosomes, small morphology and the like, and novel techniques and methods are provided for identification and structural research on crambe cordifolia chromosomes, evolution research on crambe cordifolia, and the like.