The capability of 3D printing technologies for direct production of complex 3D structures in a single step has recently attracted an ever increasing interest within the field of microfluidics. Recently, ultrafast lasers have also allowed developing new methods for production of internal microfluidic channels within the bulk of glass and polymer materials by direct internal 3D laser writing. This review critically summarizes the latest advances in the production of microfluidic 3D structures by using 3D printing technologies and direct internal 3D laser writing fabrication methods. Current applications of these rapid prototyped microfluidic platforms in biology will be also discussed. These include imaging of cells and living organisms, electrochemical detection of viruses and neurotransmitters, and studies in drug transport and induced-release of adenosine triphosphate from erythrocytes.