A tri-signal ultrasensitive colorimetric/electrochemical detection of ovomucoid (OM) was developed. Herein, copper oxide nanoparticles (CuO NPs) were prepared, which exhibit excellent enzyme-like activity (peroxidase-like and laccase-like) and electrochemical activity. CuO@3-APBA nanoparticles (CuO@3-APBA NPs) were prepared by the coordinating Cu with the amino group on 3-aminophenobenic boric acid (3-APBA) in CuO NPs. 3-APBA of CuO@3-APBA can react with diol structure on sugar chain of OM under alkaline conditions. Then, a tri-signal ultrasensitive biosensing platform for OM was established based on the catalytic activity of CuO@3-APBA nanozyme. For the first signal, CuO@3-APBA can catalyze oxidation of 1,3,5-trimethylbenzene (TMB) to turn the solution from colorless to blue in the presence of H2O2 (absorbance at 652 nm). For the second signal, CuO@3-APBA can catalyze the oxidation of substrates (2, 4-dichlorophenol and 4-aminoantipyrine) and turn the solution from colorless to pink (absorbance at 510 nm). For the third signal, electrochemical oxidation peak of copper ion from Cu+ to Cu2+ of CuO@3-APBA was recorded by differential pulse voltammetry, which was used to determine the OM. The sensing platform exhibited a wide linear range (0.0000316–100.000000ng/mL) with a low detection limit (0.0105 pg/mL), as well as showed advantages, such as satisfactory reproducibility, good stability, and excellent selectivity. The assay has the potential applications for ultrasensitive detection of allergen in foods.