Abstract: To improve the immunoassay performance of mycotoxin anti-idiotype nanobody and to realize mimicry of bispecific detected antigens, in this study, anti-idiotype nanobody against zearalenone (ZEN) and deoxynivalenol (DON) were used, and the SpyCatcher/SpyTag tags were fusion-expressed with N28 and Z6, respectively, and based on the in vitro self-assembly, the construction study of bivalent and bispecific anti-idiotype nanobody of N28 and Z6 were carried out. The results showed that the prokaryotic soluble expression of four SpyCatcher/SpyTag labeled fusion anti-idiotype nanobody, N28-SpyCatcher, N28-SpyTag, Z6-SpyCatcher, and Z6-SpyTag were achieved. The IC₅₀ of the competition inhibition standard curves established based on Z6-SpyCatcher and Z6-SpyTag were 0.18 and 0.20 ng/mL, respectively, and their sensitivities were significantly higher than that of the IC₅₀ of monomeric Z6 (0.28 ng/mL). The fusion of the SpyCatcher tag with N28 was favorable for the enhancement of the sensitivity of the N28 assay, with the IC₅₀ value decreased from 94.39 to 42.33 ng/mL, whereas the fusion of SpyTag label with N28 inhibited its binding to DON monoclonal antibody. In addition, bivalent and bispecific anti-idiotype nanobody Z6-Z6, N28-28, and N28-Z6, Z6-N28 of Z6 and N28 were successfully prepared based on the in vitro self-assembly property of SpyCatcher/SpyTag under the simple reaction condition of 4 ℃ for 12 h. The characterization results of the ELISA showed that the bivalent anti-idiotype nanobody retained their binding properties to the corresponding antibodies. The binding properties of the corresponding antibodies, in which N28-Z6 also achieved the binding activity with ZEN and DON antibodies simultaneously, and Z6-N28 only retained the binding activity with ZEN antibody because the binding of SpyTag tag to N28 would hinder its binding to DON antibody. In conclusion, the present study realized a simple and rapid preparation of ZEN/DON bivalent and bispecific anti-idiotype nanobody based on SpyCatcher/SpyTag and suggested that the SpyCatcher tag had a facilitating effect on the immunoassay performance enhancement of ZEN/DON anti-idiotype nanobody.