The carboxyl groups of poly(acrylic acid) (PAA)/polyvinyl alcohol (PVA) microfibrous membranes were activated by using dicyclohexyl-carbodiimide and N-hydroxysuccinimide. The free amines of glucoamylase were reacted with the activated carboxyls to form ester bond, which immobilized the enzyme onto the electrospun PAA/PVA microfiber by covalent bonds. The enzymatic reaction of the immobilized glucoamylase was investigated. The results showed that glucoamylase was attached on the surface of electrospun PAA/PVA composite microfibrous membrane, and the diameter of the fiber was about 435 nm. The maximum reaction velocity (Vmax) and michaelis constant (Km) of the immobilized glucoamylase were 5.77 g/(L×h) and 10.45 g/L, respectively. The optimum pH range and temperature of the immobilized enzyme were similar to those of the free enzyme. Immobilized glucoamylase had higher storage stability. It retained 62% of the original activity in 12 d, as compared to about 40% for the free enzyme. The immobilized glucoamylase could be used for 10 times while retaining 40.5% of its activity.