Reversible‐addition‐fragmentation chain transfer polymerization method was adopted here in this study to prepare a series of thermo‐sensitive tri‐block copolymer carrier, poly(N,N‐diethylacrylamide‐b‐β‐hydroxyethyl methacrylate‐b‐glycidyl methacrylate), PDEA‐b‐PHEMA‐b‐PGMA (named as DHG). Their structures and temperature sensitivity performances were further characterized by the nuclear magnetic resonance, the gel permeation chromatography, and the fluorescence spectroscopy, respectively. It has been identified that the synthesis reaction of tri‐block copolymer was living polymerization. The thermo‐sensitivity study suggested that 2 monomers, β‐hydroxyethyl methacrylate and glycidyl methacrylate, played key roles on the lower critical solution temperature performance. Finally, above block copolymer was conscripted to immobilize penicillin G acylase; relationships among the number‐average molar weight
()trueM¯n of the 2 monomer, β‐hydroxyethyl methacrylate and glycidyl methacrylate, loading (L), and activity recover rate (Ar) of immobilized PGA were investigated. And the results showed that L and Ar arrived at 19 502 U and 92%, respectively.