Potential ecological environmental and food safety risks of various Cry toxins of Bacillus thuringiensis (Bt) in transgenic food have received gradually increasing attention, which urged to establish an efficient and broad-spectrum detection technology for Cry toxins. Based on the single-domain antibody (sdAb) A8 against Bt Cry1Ab toxin screened from the humanized domain antibody library, the key amino acids of sdAb (A8) binding five kinds of Cry1 toxins were predicted using homology modeling and molecular docking technology, and the results showed that 105th asparagine, 106th arginine, 107th valine, and 114th arginine, respectively, located in heavy-chain complementarity-determining region 3 were common key amino acid sites. Subsequently, site-saturation cooperative mutagenesis of the four key sites was performed using overlap extension PCR, and multiple site-saturation mutagenesis sdAb library with the capacity of 1.2 × 105 colony-forming units (CFU) was successfully constructed. With alternating five Cry1 toxins as coating antigen, two generic sdAbs (2-C1, 2-C9) were screened out from the mutagenesis library, which could detect six kinds of Cry1 toxins at least. Through ELISA analysis, the binding activity of 2-C9 was significantly enhanced, and its OD values versus Cry1Aa, Cry1Ab, Cry1B, Cry1C, and Cry1E increased to 1.34, 1.53, 1.82, 2.39, and 2.7 times, respectively, compared with maternal antibody A8. The IC50 values of 2-C9 against Cry1Aa, Cry1Ab, Cry1B, and Cry1C were lower than that of A8, which showed that the affinity of 2-C9 against Cry1 toxins was enhanced. The results were beneficial to developing high-throughput and high-sensitive immune-detecting technology for Cry toxins.