3D printing is an emerging enabling technology that can facilitate the production of complex 3D structures in analytical chemistry, including the millifluidic, e.g. flow injection (FI), and microfluidic arenas. In this review, the potential of 3D printing for the fabrication of cost-effective millifluidic platforms incorporating on-line sample handling and separation is critically appraised against traditional configurations or manufacturing processes. Applications resorting to structures achievable with 3D printing, in some instances exploiting the surface chemistry of the printable material, are grouped under: (i) fluidic drivers, mixers and reactors, (ii) membrane separation, (iii) sorbent extraction/concentration, (iv) chromatographic and electrophoretic separation, and (v) sensing and detector housings. Summary tables are also presented for reported applications of on-line sample handling and separation in environmental and biochemical analysis.