The introduction of foreign cargo into living cells with high delivery efficiency and cell viability is a challenge in cell biology and biomedical research. Here, we demonstrate a nanosecond pulse laser-activated photoporation for highly efficient intracellular delivery using titanium dioxide (TiO2) microspikes as a substratum. The TiO2microspikes were formed on titanium (Ti) substrate using an electrochemical anodization process. Cells were cultured on top of the TiO2microspikes as a monolayer, and the biomolecule was added. Due to pulse laser exposure of the TiO2microspike-cell membrane interface, the microspikes heat up and induce cavitation bubbles, which rapidly grow, coalesce and collapse to induce explosion, resulting in very strong fluid flow at the cell membrane surface. Thus, the cell plasma membrane disrupts and creates transient nanopores, allowing delivery of biomolecules into cells by a simple diffusion process. By this technique, we successfully delivered propidium iodide (PI) dye in HeLa cells with high delivery efficiency (93%) and high cell viability (98%) using 7 mJ pulse energy at 650 nm wavelength. Thus, our TiO2microspike-based platform is compact, easy to use, and potentially applicable for therapeutic and diagnostic purposes. © The Royal Society of Chemistry 2021.