Staphylokinase (SAK) is a bacterial protein with profibrinolytic activity. However, SAK suffers from short serum half-life and high immunogenicity. PEGylation with high Mw (20 kDa or 40 kDa) could decrease the immunogenicity and prolong the serum half-life of the proteins. However, the PEGylated protein could induce the anti-PEG antibodies and its bioactivity was significantly decreased. Arabinogalactan (AG) is a health-promoting substance with numerous biological activities. Conjugation of AG is an alternative strategy to solve the above-mentioned problems. However, conjugation with AG significantly decreased the bioactivity of a protein by shielding the bioactive domain. Here, AG conjugation and PEGylation were combined to improve the therapeutic efficacy of SAK. PEG with low Mw (2 kDa or 5 kDa) acted as a linker to conjugate AG from Larix. As compared with SAK-AG (22.3%), the conjugates (SAK-P2K-AG and SAK-P5K-AG) largely maintained the bioactivity of SAK (73.8% and 62.9%). The two conjugates both showed an 8-fold decrease in the SAK-specific IgG titers and a prolonged serum half-life. Moreover, the conjugates did not render any apparent toxicity to the heart, liver and renal functions of mice. Thus, our conjugation strategy is promising for the development of an effective long-acting therapeutic protein. (C) 2019 Elsevier B.V. All rights reserved.