Thermostabilization of L-α-glycerophosphate oxidase (GPO) by chemical modification with oxidized dextran was performed. The enzyme was coupled with oxidized dextran produced by periodate oxidation, followed by reduction with sodium cyanoborohydride and glycine blocking. The modified enzyme (GPO-Dextran-Gly) thus obtained showed high resistivity against heat treatment compared with that of native GPO, though the residual activity after chemical modification was relatively low. In order to get modified enzymes with higher residual activity after modification, the repression of inactivation of GPO during the coupling reaction and the dialysis procedure was investigated. The addition of 10 mM flavine adenine dinucleotide (FAD) could repress the inactivation during the coupling reaction. Furthermore, dialysis of the coupled enzyme against a buffer solution containing 10% ammonium sulfate reduced inactivation during the dialysis procedure. After native GPO was heated at 50℃ for 60 min in an incubator, the activity of GPO was reduced to 9.6% from the original (native 100%). On the other hand, the chemical modification with oxidized dextran itself reduced the GPO activity to 47.7% (A) from the original (native 100%). The modified enzyme (GPO-Dextran-Gly), however, acquired heat resistivity, and remamined its activity up to 78.3% (B) under the same heat treatment. Thus, the overall residual activity converted from the original activity (A×B) was calculated as 37.3%. The GPO-Dxtran-Gly was applied to the determination of L-α-glycerophosphate with a good applicability.