Several strategies are currently employed to enhance the detection limit of bead-based assays, but all these approaches improve the sensitivity by varying the assay procedures chemically or biologically. In previous digital microfluidic setups for bead-based immunoassay, the magnetic beads were suspended for detection. We investigated the effect of bead aggregation in such an immunoassay system and propose the aggregation magnified post-assay fluorescence for ultrasensitive immunodetection on digital microfluidics (AMPFLUID). The detection signal and sensitivity are further enhanced even at the post-assay stage without altering the original assay protocol on employing magnetically triggered post-assay aggregation of beads in a digital microfluidic setup followed by processing of the fluorescent signal. This method is shown to enhance the fluorescent signal with increased consistency and sensitivity after appropriate charge-coupled device (CCD) calibration. This method of on-chip detection allows the fulfilment of consumption of a volume at the nanoliter level and a limit of detection in the range picogram/mL. In our sTNF-RI model immunoassay, only 2.5 nL of sample is required; a detection limit 15 pg/mL is achieved. The decreased uncertainty of the measure is indicated by the error bars and coefficient of variation.