Microfluidics offers transformative potential in healthcare by enabling miniaturized, user-friendly, and cost-effective devices for disease diagnostics among other biomedical applications; however, their meaningful adoption is severely hindered, especially in developing countries and resource-limited settings, by the cost, time, and complexity of their fabrication. To overcome this barrier of access, this work develops a novel approach for highly efficient (<4 h), cost-effective, and clean-room-free fabrication of functional polydimethylsiloxane (PDMS)-based microfluidic devices based on coupling stereolithography three-dimensional (3D) printing with hot embossing. The strategy exhibits high fidelity between the digital design and final device, remarkable transfer accuracy between the 3D print and poly(methyl methacrylate) (PMMA) mold, in addition to highly smooth surfaces (Ra < 1 μm). To establish the versatility of the approach and performance quality of the fabricated devices, three advanced microfluidics-driven biosensing platforms are developed: a microsphere droplet generator, a stop-flow lithography-based hydrogel microparticle synthesizer, and a hydrogel postembedded microfluidic device for multiplexed biomarker detection. As a proof-of-concept, the latter platform was applied to the multiplexed detection of microRNA, a highly promising class of liquid biopsy biomarkers for many diseases including cancer. Notably, the ability to demonstrate multiplexed sensing of disease biomarkers within devices made through a facile, rapid, and clean-room-free strategy demonstrates the immense potential of this fabrication approach to accelerate the adoption and advancement of biomedical microfluidic devices in practice and in resource-limited settings.