The Drosophila intestine is a powerful model for stem cell dynamics and epithelial biology, yet no intestinal derived cell lines have been available until now. Here, we describe the establishment of Drosophila intestinal cell lines. The cell lines were derived from the embryonic intestine through specific Ras expression and show continuous proliferation and the ability to be frozen and re-thawed. Each derived cell line exhibited morphological and cellular heterogeneity. Single-cell RNA sequencing confirmed their intestinal origin and cell populations with unique enriched signaling pathways. In addition, L15, one of the three lines, formed 3D spheroids that displayed epithelial polarity. Together, these lines provide an additional resource for studying intestinal development, epithelial organization, and pest management.