As they are widely implemented as features formed at a continuous metal surface, Surface-enhanced Raman spectroscopy (SERS) substrates are amenable to implementation in a microfluidic format. However, achieving reproducibility and repeatability in platform fabrication often requires methods that are complex and expensive, restricting transfer from laboratory prototyping to mass production. Herein, a new and robust, low cost technique for cavity array SERS substrate fabrication and its integration into a microfluidic device is described. The fabrication relies on 2-photon 3D printing of a master template and enables batch fabrication of reproducible arrays with sub-micron resolution and uncomplicated modification of the cavity architecture. With this approach, significant Raman signal enhancement of up to 6.7 × 107 through incorporation of nanostructures - plasmonic “hot-spots” and straightforward integration into microfluidic device was achieved. In addition, implementing the nano-sub-structuring to the cavities by 2-photon 3D printing approach, we obtain reproducible enhancements of SERS intensity of more than 4 fold in comparison to arrays fabricated using previously established sphere templating techniques. The fabricated microfluidic device showed detection capability for cholesterol analyte in solution in a simple demonstration where the array was modifed with thiolated cyclodextrin host to enable analyte capture.