High-throughput and rapid arsenite (As(III)) monitoring is an urgent task to deal with the critical threat from As(III) contamination in the environment. In this study, an effective, portable, and sensitive As(III) assay was developed using the plasmonic silver (pAg) chips for As(III) detection. The pAg chips were fabricated by a simple seed-mediated method to grow the silver nanoisland films (Ag-NIFs) with the compact nanoislands and adjustable interisland gaps on the large-sized substrates. With appropriate surface functionalization and optimal chip manufacturing, Cy7.5 fluorescence dye can be immobilized on the surface of Ag-NIFs in the presence of As(III) to output the enhanced fluorescence signals up to 10-fold and improve the detection limit of As(III) less than 10 ppb. According to our results, the high-throughput detection measurements and wide dynamic range over 4 orders of magnitude implied the broad prospects of pAg chips in fluorescence-enhanced assays. The proposed As(III) assay has shown great opportunities for the practical application of ultratrace As(III) monitoring.