Spatial patterning of biomolecules on a surface with nano- and micrometer precision is important in engineering biological microenvironments. Nano- and micropatterned biological arrays are useful for chemical functionalization of miniaturized biosensors, tissue engineering, high throughput drug screening, and fundamental biophysical and molecular biology studies. Despite the growing demands and applications, current surface patterning techniques are still lacking. Parylene (or poly[p-xylylene]) is a unique family of chemically vapor deposited polymer that is biocompatible, exhibits little to no swelling in aqueous solutions and is amenable to photolithography processing. These characteristics of parylene are attractive for its use as a "peel-able", high fidelity stencil for the surface patterning of biomolecular arrays. Different types of parylene coatings containing substituted functional groups have been developed for biochemical surface modification on virtually any surface. The two themes that emerge from this work are: i) the use of parylene films with nano/microfabricated openings as a stencil tool for surface patterning (Chapters 2 - 4), and ii) the application of parylene coatings to directly pattern surfaces (Chapters 5 and 6). Chapter 2 describes a new nanofabrication process to create parylene stencils with sub-100nm openings. These stencils can then be combined with inkjet printing to rapidly generate multi-component biomolecular nanoarrays on a single surface. The work in Chapter 3 elucidates the role of cell-cell interactions in tumor angiogenesis through the use of patterned tumor cell arrays. In Chapter 4, a hybrid substrate, consisting of a hydrophobic parylene stencil with hydrophilic silanized openings, is utilized to control spot drying morphology and improve microarray reproducibility. Parylene-based paper microfluidic devices and their enhanced capabilities are presented in Chapter 5. The work in Chapter 6 involves proteins covalently immobilized onto an array of reactive (aminated) parylene strips. These strips subsequently incorporated with microfluidics for the affinity-based screening and subsequent recovery of aptamers. Chapter 7 is a summary containing unpublished observations of parylene that may serve as a useful starting point for future researchers. These cumulative studies demonstrate that parylene-based surface patterning techniques are versatile for creating nano- and microarrays towards many biological applications.