Abstract Simultaneous identification of multiple nucleic acid targets is pivotal for high-throughput analysis in clinical diagnostics. Unfortunately, conventional PCR offers limited multiplexing capability due to issues like primer interference, fluorescence spectral overlaps and instrumentation complexity. Solid-phase PCR (SP-PCR), in which primers are physically separated on a solid support, has emerged as an alternative strategy for detection of multiple targets in parallel. However, SP-PCR has been suffering from low efficiency, long reaction time, inability for real-time signal monitoring and accurate quantification, which greatly restricts their applications in multiplexed assays. In this study, we presented for the first time a compact and portable flow-through SP-PCR platform to permit rapid, highly efficient and quantitative solid-phase amplification. We designed an integrated platform comprising a novel mechanically actuated valving system and a dual-chamber SP-PCR system to enable automated sample purification and amplification. Active oscillating the PCR solution between the chambers significantly enhanced the mass diffusion over the solid-phase array. Moreover, after each amplification cycle, the PCR solution was separated from the solid-phase-bound probes, thereby eliminating background signal interference and allowing for real-time monitoring of SP-PCR signals without the need for complex optical systems. The flow-through SP-PCR system demonstrated quantitative detection of five viral pathogens in a single reaction, with a limit of detection of 10 copies per reaction within 20 minutes. This platform provides a promising high-throughput, low-cost and simple-instrumentation multiplexed diagnostic system.