A novel surface-enhanced Raman scattering (SERS) sensor is developed for real-time and highly repeatable detection of trace chemical and biological indicators. The sensor consists of a polydimethylsiloxane (PDMS) microchannel cap and a nanopillar forest-based open SERS-active substrate. The nanopillar forests are fabricated based on a new oxygen-plasma-stripping-of-photoresist technique. The enhancement factor (EF) of the SERS-active substrate reaches 6.06 × 106, and the EF of the SERS sensor is about 4 times lower due to the influence of the PDMS cap. However, the sensor shows much higher measurement repeatability than the open substrate, and it reduces the sample preparation time from several hours to a few minutes, which makes the device more reliable and facile for trace chemical and biological analysis.