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Natural exosomes can express specific proteins and carbohydrate molecules on the surface and hence have demonstrated the great potentials for gene therapy of cancer. However, the use of natural exosomes is restricted by their low transfection efficiency. Here, we report a novel targeting tLyp-1 exosome by gene recombinant engineering for delivery of siRNA to cancer and cancer stem cells. To reach such a purpose, the engineered tLyp-1-lamp2b plasmids were constructed and amplified in Escherichia coli. The tLyp-1-lamp2b plasmids were further used to transfect HEK293T tool cells and the targeting tLyp-1 exosomes were isolated from secretion of the transfected HEK293T cells. Afterwards, the artificially synthesized siRNA was encapsulated into targeting tLyp-1 exosomes by electroporation technology. Finally, the targeting siRNA tLyp-1 exosomes were used to transfect cancer or cancer stem cells. Results showed that the engineered targeting tLyp-1 exosomes had a nanosized structure (approximately 100 nm) and high transfection efficiency into lung cancer and cancer stem cells. The function verifications demonstrated that the targeting siRNA tLyp-1 exosomes were able to knock-down the target gene of cancer cells and to reduce the stemness of cancer stem cells. In conclusion, the targeting tLyp-1 exosomes are successfully engineered, and can be used for gene therapy with a high transfection efficiency. Therefore, the engineered targeting tLyp-1 exosomes offer a promising gene delivery platform for future cancer therapy., Graphical abstract The tLyp-1-lamp2b plasmid transfected HEK293T cells can secreted tumor targeting tLyp-1 exosomes. By electroporation technology, targeting tLyp-1 exosomes were loaded with siRNA. When targeting tLyp-1 exosome ruptured in cytoplasm, siRNA was loaded into the RNA-induced silencing complex (RISC). The sense (passenger) strand was degraded while the antisense (guide) strand directs RISC to mRNA that has a complementary sequence, thereby resulting in the silence of target gene.Image, graphical abstract