The capture of circulating tumor cells (CTCs) is of paramount importance for early cancer diagnosis. Herein, we report the development of hyaluronic acid (HA)-immobilized electrospun random or aligned polylactic acid (PLA) nanofibers for cancer cell capture applications. Electrospun random or aligned PLA nanofibers were modified with HA via polyethylenimine (PEI) as a linker, followed by acetylating the remaining PEI amines on the fiber surface. The formed HA-immobilized random or aligned PLA nanofibers were thoroughly characterized. We show that the fiber morphology does not significantly change after multiple steps of surface modification. In addition, the HA-modified random or aligned PLA nanofibers display good hemocompatibility. Moreover, the modification of HA increases the capture capability of electrospun nanofibers towards CD44 receptor-overexpressing cancer cells, and electrospun nanofibers with aligned structure display the highest cancer cell capture effciency. The developed HA-immobilized electrospun aligned PLA nanofibers may be applied for capturing CTCs in the future after further materials design and optimization.