Electrospinning with subsequent photopatterning of copolymers containing thiol-reactive maleimide groups was utilized to fabricate micropatterned nanofiber arrays amenable for biomolecular immobilization and detection. Bead-free uniform nanofibers were obtained by electrospinning of copolymers composed of poly(ethylene glycol) methacrylate, methyl methacrylate, and maleimide based monomers. While the poly(ethylene glycol) based monomer provides necessary hydrophilicity to impart the fibers with antibiofouling properties, the methyl methacrylate component improves fiber formation. The maleimide functional group in the polymers serves a dual role of inducing photochemical cross-linking as well as enabling efficient functionalization of nanofibers using the thiol-maleimide coupling reaction. The maleimide based fiber cross-linking also enables fabrication of micropatterned arrays using photolithography. Obtained nanofiber based micropatterns undergo facile functionalization with thiol-containing dyes, protein binding ligands, and dye-labeled oligonucleotides. Hybridization studies on the oligonucleotide immobilized arrays with fluorescently labeled complementary sequence demonstrated that sensing on this platform was achievable with high specificity. Thus, efficient bioconjugation as well as detection of bioanalytes such as proteins and oligonucleotides can be undertaken on these nanofibers arrays. The strategy to obtain nanofiber based micropatterns disclosed here provides an access to a versatile interface adaptable for biomedical applications.