<p><i>Burkholderia glumae</i> (<i>B. glumae</i>) and <i>Burkholderia plantarii</i> (<i>B. plantarii</i>) are primary causal agents of rice bacterial panicle blight (RBPB) and cause substantial yield losses in rice worldwide. Given their seed-borne transmission characteristics, quarantine status and destructive hazards, rapid, super-sensitive, highly specific on-site detection technologies are urgently needed. Here, using <i>B. glumae</i> Os48 and <i>B. plantarii</i> ZJ171 as immunogens, we prepared two highly specific and ultra-sensitive monoclonal antibodies (mAbs) against <i>B. glumae</i> (4A7 and 8C5) and two highly specific and ultra-sensitive mAbs against <i>B. plantarii</i> (12B5 and 14B3). We then developed dot-enzyme-linked immunosorbent assays (Dot-ELISA) and colloidal gold immunochromatographic strip (CGICS) assays for detecting <i>B. glumae</i> and <i>B. plantarii</i> with the prepared mAbs as the detection antibodies. These developed mAb-based serological techniques enabled the rapid, broad-spectrum, and specific detection of <i>B. glumae</i> and <i>B. plantarii</i>, respectively, and showed no cross-reaction with other control plant bacteria included in the analysis. Moreover, the detection limits of the Dot-ELISAs for <i>B. glumae</i> or <i>B. plantarii</i> were up to 1.96 × 10⁴ colony-forming units (CFU)/mL, and CGICSs could detect <i>B. glumae</i> and <i>B. plantarii</i> at concentrations as low as 9.78 × 10³&#xa0;CFU/mL. Surprisingly, these two serological techniques are 2 ~ 8 times more sensitive than conventional polymerase chain reaction (PCR). Collectively, the two newly developed serological techniques in this work provide two simple, rapid, broad-spectrum, highly specific, ultra-sensitive and on-site means to detect <i>B. glumae</i> and <i>B. plantarii</i> in rice grains and leaves, thereby offering reliable and practical tools for the detection and quarantine of these two RBPB pathogens.</p>