The application of microfluidics for largescale rapid analytics holds great promise in the pharmaceutical diagnostics and analytical chemistry. Here we report a paper based microfluidic substrate designed by the impregnation of Silver nanoparticles. This study demonstrates the achievement of a thousand-fold increase in the successful detection level up to 10 ppb (90.8 nM) by employing Surface Enhanced Raman Spectroscopy (SERS) for the detection of Catechol. The presented sensor exhibits the following main features: (i) high specificity of enzyme (Hemocyanin)-based sensing, (ii) effective SERS sensitivity, (iii) easiness and cost-effectiveness of a paper-based platform. We rationalize these findings using the ab initio DFT simulations using Gaussian 09 whose theoretical calculations reflect the observed experimental Raman peaks.