Biophotonics is an exciting and fast-expanding frontier which involves the fusion of advanced photonics and biology. It has not only created many novel methodologies for biomedical research, but also achieved many significant results as an independent field. Thanks to femtosecond (fs) laser technologies, important progresses have been made regarding the manipulation, imaging, and engineering of biological samples ranging from single molecules to tissues in the last 20 years. The ultrashort pulses at near-infrared band provide many advantages: high nonlinear efficiency, low absorption by biological samples, high spatial and temporal resolution and confinement, and low phototoxicity. They are noninvasive and easy to control. Although the mechanism of how fs laser pulses interact with cells remains unclear, experimental results have shown that they could open up the cell membrane and hence made optical transfection and optical cell fusion possible. In this review, some of the seminal works on transfection and cell fusion by fs lasers are presented. The ideas behind and the experimental details will be described together with a highlight on their significances. Specifically, the thermal effect is analyzed based on multiphoton excitation and plasma formation in an aqueous environment to explain the nontoxic characteristic of fs laser irradiation. Last, some applications of fs laser induced transfection and cell-cell fusion with potential major impact in biomedical sciences are proposed.