Soluble human tumor necrosis factor receptors (shTNFRI and shTNFRII) are antagonists of tumor necrosis factor-alpha (TNF-alpha) and are under clinical investigation as therapy for autoimmune diseases and transplant rejection. However, shTNFRI and shTNFRII are limited for clinical usage because of their short half-lives in vivo. Recombinant TNF-alpha receptors (infliximab and etanercept) are used in treatment of rheumatoid arthritis and Crohn's disease but are also being tested for a number of other autoimmune diseases. Human serum albumin (HSA) has been used to construct long-acting fusion proteins. Here, we report the effect of fusion of HSA with shTNFRI and with shTNFRII on shTNFR's neutralizing activity against TNF-alpha. HSA fusion proteins were separately expressed in Pichia pastoris. Purified recombinant shTNFRI-HSA, HSA-shTNFRI and HSA-shTNFRII could block the cytolytic activity of TNF-alpha in L929 cells, and the fusion at N-terminus of shTNFRI could result in larger degree of activity decline than that at the C-terminus. Activity of three fusion proteins was much weaker than etanercept, which demonstrated that fusion of HSA significantly influenced TNF-alpha neutralizing activity of shTNFRs. Compared with Fc fragment, HSA fusion technology may therefore not be an ideal strategy in development of long-acting shTNFRs protein drugs.