Drosophila melanogaster is a widely used model organism in many biological studies. Usually D. melanogaster inbreeding lines maintained in lab are with a high density, therefore one line might be invaded by other lines. Up till now, there is no effective way to check the line admixture in a large scale. In this study, we serve a new method, Line-seq, to solve this problem by taking advantage of both targeted sequencing and barcoding sequencing techniques. Using Line-seq, we successfully detected line admixture in 23 inbreeding lines and compared the performance of Line-seq and traditional Sanger sequencing in eight artificially admixed samples. Line-seq can detect exogenous DNA admixtures as low as 6%, while Sanger sequencing can do so when the proportion of of exogenous DNA rises up to around 20%. Our results highlight Line-seq is an sensitive, accurate, as well as cost-effective method to detect D. melanogaster line admixture.