This study reports development of a microfluidic device for highly sensitive and selective detection of a β-amyloid peptide (Aβ1-42) in simulated cerebrospinal fluid, using surface-enhanced Raman spectroscopy (SERS). The device ensemble comprises a purine ligand (Pu) and its interaction with silver nanoparticles (AgNPs) to generate SERS hotspots. The low surface energy of the synthesized Pu ligand and high surface energy of AgNPs are utilized for the functionalization and formation of a Pu-AgNP SERS substrate. We have integrated a novel polydimethylsiloxane (PDMS) microfluidic device with Pu-AgNPs using a combination of photo- and soft lithography fabrication, sealed by thermal cross-linking with another layer of PDMS, to produce an effective screening platform for Aβ1-42. The SERS spectrum from the microfluidic device affords almost noise-free measurements, with excellent limit-of-detection values.