In the field of life sciences, the monitoring of biological samples has become a great concern to control the ecosystem evolution. However, their characterization is often time-consuming because the typical size of the organisms/particles of interest is several orders of magni- tude smaller than the size of the sample under observation. Optical visualization systems require, then, high magnifi- cations that severely limit the depth of focus and conse- quently decrease the sampling rate. To tackle this issue, the most straightforward technique consists in focusing the samples to fit the observation field of view by means of so- called ''sheath flows''. This expedient allows for increasing the overall flow rate, inversely related to the sampling time. In this article, a cost-effective 3D hydro-focusing device is presented. Several flow rates have been tested for both sample and sheath flows, and a thorough investigation of the shape of the focused streamlines conducted in order to validate the prototype design. The 3D position of the sampled micro-objects has been located by digital holo- graphic microscopy and their distribution in cross-sections downstream the injection nozzle compared to numerical simulations. A maximum constriction—ratio between the part of the cross-sections where particles are present with and without focusing sheath flow—of 4.4 % has been observed confirming the potentiality of the technique. Also, a successful match between experiment and numerical simulation has been noted.