PURPOSE: To simply and rapidly regenerate or exchange the subject substrate by treating an enzyme activity measuring filler comprising a biotinylated substrate bonded to an avidin-bonded support to remove the biotinylated substrate, and subsequently immobilizing a new biotinylated substrate on the remained avidin-bonded support.

CONSTITUTION: When the substrate of an enzyme activity measuring filler in which a biotinylated substrate comprising biotin bonded to a substrate recognized by the enzyme is immobilized on a water-insoluble avidin-bonded support by the affinity between the avidin and the biotin is regenerated, the affinity between the avidin and the biotin is weakened to remove the immobilized biotinylated substrate from the avidin-bonded support. A new biotinylated substrate recognized by the enzyme is subsequently bonded to the remained avidin- bonded support. In order to weaken the affinity between the avidin and the biotin for the removal of the biotinylated substrate, for example, the filler for measuring the activity of the enzyme is brought into contact with a solvent solution of a modifying agent such as guanidine hydrochloride.