This work presents the synthesis of anisotropic gold nanocrystals (Au NCs) using a segmented flow-based microfluidic device, and intracellular delivery using the synthesized particles. The device consists of T and Y shape junctions, cross channel, and winding geometries, where reagents mix. Reagents meet at the initial Y-junction and the T-shape junction generates droplets. Synthesized Au NCs are characterized and are found to possess multiple plasmonic peaks as per their size, shape and anisotropic nature. These plasmonic peaks can be tuned to the near-infrared region, which is advantageous in different biomedical applications. With the synthesized Au NCs as a mediator, propidium iodide dye is successfully delivered into the cellular cytoplasm of HeLa cells by using nanosecond pulse laser. The results demonstrated that Au NCs synthesized using a segmented flow-based microfluidic device can be used for the intracellular delivery of different exogenous molecules. The best results are achieved as 96% delivery efficiency and 98% cell viability. Thus, our platform potentially applicable to anisotropic Au NCs synthesis as well as cellular therapy and diagnostic purpose. © 2020 IEEE.