The invention relates to an artificial microsphere for detecting a rice and cowpea trypsin inhibitor. The artificial microsphere for detecting the rice and cowpea trypsin inhibitor is coated by an anti-CpTI protein polyclonal antibody, the microsphere particle size is 200nm, and a surface carrying group is carboxyl. The artificial microsphere is prepared by the following steps: according to a preferable codon expressed by prokaryotic cells, remodifying and establishing a whole-length CpTI gene expression sequence, establishing a germ fusion expression carrier, performing restriction enzyme cutting and insertion into a glutathione-S-transferase (GST) gene fusion expression carrier, performing bacterium expression, further purifying by using an affinity chromatography column so as to obtain a GST-CpTI fusion protein, further dissolving an adjuvant with an antigen, immuning mice, performing separation purification so as to obtain the anti-CpTI protein polyclonal antibody, and performing coupling activation on the antibody by using a chemical coupling method, thereby obtaining artificial microspheres for detecting the CpTI protein. The artificial microsphere is capable of detecting the lowest detection limit of 0.2mu g/ml of the CpTI fusion protein, and a correlation coefficient R2 is 0.67.