Electrohydrodynamic (EHD) jet printing is a direct-writing technique which ejects ink through a fine nozzle using an electric field, which has the advantages of high-resolution, rapid printing speed and a wide range of ink selectivity. In this article, the EHD jet printing system is utilized to print patterns of polystyrene (PS) nanofibers. The effect of parameters such as ink concentration, working distance, applied voltage, and stage speed on the diameter of the printed nanofibers was investigated. The EHD jet printing technology is further utilized to print various patterns of polydiacetylene (PDA)-embedded PS nanofibers. The EHD jet printing based nanofiber printing is advantageous over conventional electrospining based approaches in terms of patterned PDA images. In addition, an advanced EHD jet printing system which is adopted for aligned nanofiber printing will expand the application of nanofibers from bio and chemical sensors to tissue engineering and electronics.