Cyanogen bromide has been widely used as an activator to some resins while preparing affinity chromatography column and immobilized enzymes. Nevertheless, it is a hazard poison with a volatilization feature, which should be carefully used. Here, 1,1′-carboxyl-diimidazole is used as an activator to Sepharose CL-6B, in order to immobilize earthworm fibrinlytic enzyme Ⅲ-1(EFE-Ⅲ-1), m-aminophenylboronic acid and to detect glycated D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH), with a low poison, a high coupling rate and a convenient process.