PEGylation can effectively improve the therapeutic potential of staphylokinase (SAK), a thrombolysis agent for therapy of myocardial infarction. However, polyethylene glycol (PEG) can sterically shield SAK and drastically decrease its bioactivity. In the present study, N-terminally PEGylated SAKs (5 and 20kDa PEG), C-terminally PEGylated SAKs with phenyl linker and the ones with amyl linker (5 and 20kDa PEG) were prepared. The effects of the PEG length, the PEGylation site and linker chemistry on the bioactivity of the heat-treated PEGylated SAK were investigated. Heat treatment at 70°C for 2h can improve the bioactivity of the C-terminally PEGylated SAKs, where the one with amyl linker and 20kDa PEG showed the highest increase extent (27%) in the bioactivity. Thus, our study can advance the development of long-acting pharmaceutical protein with high bioactivity.