Near-field electrospinning has been demonstrated to be able to achieve direct-write and highly aligned chitosan nanofibers (CNF) with prescribed positioning density. Cell spreading in preferential direction could be observed on parallel-aligned nanofibers, and the CNF patterns were capable of guiding cell extension when the distances between them are 20 and 100 μm, respectively. Alignment of the cells was characterized according to their elongation and orientation using the fast Fourier transform data and binary image analysis. Parallel CNF indicates that the alignment values sequentially increased as a function of positioning density such that incrementally more aligned cells were closely related to the increasing CNF positioning density. These maskless, low-cost, and direct-write patterns can be facily fabricated and will be a promising tool to study cell-based research such as cell adhesion, spreading, and tissue architecture.