The unprocessed macadamia protein was extracted by alkali-soluble acid precipitation method, and the rat and rabbit polyclonal antibodies were prepared respectively. The rabbit and the murine polyclonal antibodies were used as the capture and detection antibodies respectively to establish a double-antibody sandwich enzyme-linked immunosorbent assay. The detection limit of this method was (0.95±0.17) ng/mL. The method had no obvious cross-reaction with soybean, walnut, hazelnut, almond, peanut, pistachio, cashew and wheat protein, which proved that the method was specific. The recovery rate of macadamia protein in baked macadamia nuts and baked biscuits is in the range of 73.67%～77.03%. This method was not only suitable for the accurate detection of unprocessed macadamia protein, but also for the quantitative detection of macadamia protein after hot processing and baked goods.