In this study, we report a newly developed three-dimensional (3D) biological printer using non-contact, electromechanical microvalves with a nozzle diameter of 150 μm. To control and utilize this printer for life science applications, we developed an easy-to-use control software with a graphic user interface (GUI). First, using the printer, we tested the viability of dispensed mammalian cells after printing, and there was no significant difference in viability between dispensed cells and conventionally plated cells. Next, we constructed a 3D hydrogel scaffold by printing collagen hydrogel precursor layer-by-layer with linear patterns of gelatin inside. Using the same scheme, neurons were printed and patterned in multi-layered collagen scaffold. The on-demand capability to print cells and hydrogels in multi-layered hydrogel scaffold offers flexibility in generating artificial 3D tissue composites.