Additive manufacturing, also known as three-dimensional (3D) printing, is an approach in which a structure may be fabricated layer by layer. For 3D inkjet printing, droplets are ejected from a nozzle, and each layer is formed droplet by droplet. Inkjet printing has been widely applied for the fabrication of 3D biological gel structures, but the knowledge of the microscale interactions between printed droplets is still largely elusive. This study aims to elucidate the layer formation mechanism in terms of the formation of single lines and layers comprised of adjacent lines during drop-on-demand inkjet printing of alginate using high speed imaging and particle image velocimetry. Inkjet droplets are found to impact, spread, and coalesce within a fluid region at the deposition site, forming coherent printed lines within a layer. The effects of printing conditions on the behavior of droplets during layer formation are discussed and modeled based on gelation dynamics, and recommendations are presented to enable controllable and reliable fabrication of gel structures. The effects of gelation on droplet impact dynamics are found to be negligible during alginate printing, and interfaces are found to form between printed lines within a layer depending on printing conditions, printing path orientation, and gelation dynamics.