This research aims to fabricate plane-type substrates for evaluating the axon behaviors of neuronal cells in vitro toward the development of brain-on-chip models by applying the functions of diamond-like carbon (DLC) thin film deposition, which helped to eliminate the costly and time-consuming lithography process by utilizing a shadow mask. The DLC thin films were partially deposited on stretched polydimethylsiloxane (PDMS) substrates covered with a metal mask by the plasma chemical vaper deposition method, and using the substrates culture teats with human neuroblastoma cells (SH-SY5Y) were performed. Three patterns of interconnection structures of axons were created on the substrates with disordered and regular linear wrinkle structures with several μm size formed by the depositions. The patterns were characterized by the structure that the aggregations of axons formed on the linear DLC thin film deposited areas were separately placed in regular intervals and connected each other by plenty of axons, which were individually taut in a straight line at about 100 to over 200 μm in length. The substrates expected of uses for evaluation of axon behaviors are available without fabricating guiding grooves by conventional soft lithographic methods requiring multiple stages and their treating times.