Presently, nanomanufacturing capabilities limit the commercialization of a broader range of nanoscale structures with higher complexity, greater precision and accuracy, and a substantially improved performance. Atomic force microscopy (AFM)-based nanomachining is a promising technique to address current limitations and is considered a potential manufacturing (MFG) tool for operations such as machining, patterning, and assembling with in situ metrology and visualization. Most existing techniques for fabrication of nanofluidic channels involve the use of electron-beam lithography, which is a very expensive process that requires a lengthy calibration procedure. In this work, atomic force microscopy (AFM) is employed in the fabrication of nanofluidic channels for medical applications. Channels with various depths and widths are fabricated using AFM indentation and scratching. A nanoscale channel is mainly used in the study of the molecular behavior at single molecule level. The resulting device can be used for detecting, analyzing and separating biomolecules, DNA stretching, and separation of elite group of lysosome and other viruses. The nanochannels are integrated between microchannels and act as filters to separate biomolecules. Sharply developed vertical microchannels are produced from deep reaction ion etching. Poly-dimethylsiloxane (PDMS) bonding is performed to close the top surface of the silicon device. An experimental setup is used for testing by flowing fluid through the channels. A cost evaluation shows 47.7% manufacturing-time and 60.6% manufacturing-cost savings, compared to more traditional processes.