Bead-based immunoassay has been growing as a promising technology in the point-of-care diagnostics due to great flexibility. For samples with dilute analytes, the colloidal particles can concentrate dispersed analytes and act as carriers for advanced particle manipulation. To realize a rapid in-situ immunoassay, Firster resonance energy transfer (FRET) was used herein to ensure only the target antigen lipocalin 1 (LCN1) to be detected. The measurement was made in an aqueous droplet sandwiched between two parallel electrodes. In addition, an optoelectrokinetic technique was used to further concentrate the functionalized particles to enhance the FRET fluorescent signal. The detection was usually completed within several tens of seconds. A comparison between the based-based diagnostics coupled with the optoelectrokinetic technique and a conventional ELISA reader showed nearly 100-fold improvement of the bio-signal was made. The technique is expected to bring hopes for diagnosing diseases with dilute biomarkers in the early stage.