Collective cell migration plays a pivotal role in development, wound healing, and metastasis, but little is known about the mechanisms and coordination of cell migration in 3D microenvironments. Here, we demonstrate a 3D wound healing assay by photothermal ablation for investigating collective cell migration in epithelial tissue structures. The nanoparticle-mediated photothermal technique creates local hyperthermia for selective cell ablation and induces collective cell migration of 3D tissue structures. By incorporating dynamic single cell gene expression analysis, live cell actin staining, and particle image velocimetry, we show that the wound healing response consists of 3D vortex motion moving toward the wound followed by the formation of multicellular actin bundles and leader cells with active actin-based protrusions. Inhibition of ROCK signaling disrupts the multicellular actin bundle and enhances the formation of leader cells at the leading edge. Furthermore, single cell gene expression analysis, pharmacological perturbation, and RNA interference reveal that Notch1-Dll4 signaling negatively regulates the formation of multicellular actin bundles and leader cells. Taken together, our study demonstrates a platform for investigating 3D collective cell migration and underscores the essential roles of ROCK and Notch1-Dll4 signaling in regulating 3D epithelial wound healing.