Specific targeting of tumor necrosis factor (TNF)-alpha antagonist to the inflamed site could increase its efficacy and reduce side-effects. Here, we constructed a bispecific diabody (BsDb) that targets TNF-alpha and ED-B-containing fibronectin, a fibronectin isoform specifically expressed in the pannus of the inflamed synovium in rheumatoid arthritis. BsDb was secreted from Pichia pastoris as functional protein and was purified to homogeneity. BsDb could simultaneously bind to human TNF-alpha and B-FN and neutralize TNF-alpha action. Additionally, BsDb showed a significant gain both in the antigen-binding affinity and in TNF-alpha-neutralizing ability as compared to its original antibodies, L19 and anti-TNF-alpha scFv, which were produced in E. coli. BsDb was constructed and was endowed with enhanced bioactivities and improved production processing. Therefore, it holds great potential for in vivo applications.