The expensive fabrication of current opto-microfluidic sensors is a barrier to the successful adoption of these devices in point-of-care testing. This work reports a simple inexpensive opto-microfluidic device incorporating a poly(dimethylsiloxane)-glass hybrid microfluidic chip modified with gold nanoparticles and a high-detectivity, high-stability organic photodetector. The enhancing effect of the gold nanoparticles on horseradish peroxidase-luminol-H2O2 chemiluminescence was exploited in rapid single-analyte immunoassays. The limit of detection for 17-β estradiol was 2.5 pg/ml, which is ∼200 times more sensitive than previously reported chemiluminescent immunosensors employing other organic photodetectors. Detection was also demonstrated in complex media, including natural water and blood serum.