Sandwich enzyme-linked immunosorbent assay (ELISA) microarrays have great potential for validating disease biomarkers. Each ELISA relies on the availability of two robust affinity reagents that retain activity when immobilized on a solid surface or when labeled for detection. Current procedures for the development of traditional immunoglobulin G (IgG) are slow and expensive. One alternative to IgG are single-chain antibodies (scFv), which are affinity reagents that have greater potential for high-throughput production. Unfortunately, scFv are typically less active than IgG following immobilization on a solid surface and not always suitable for use in sandwich ELISAs. We have investigated different immobilization strategies and scFv constructs to determine a more robust strategy for using scFv as ELISA reagents. Two promising strategies emerged from these studies: (1) the precapture of epitope-tagged scFv using an anti-epitope antibody and (2) the direct printing of a thioredoxin/scFv fusion protein on glass slides. Both strategies improved the stability of immobilized scFv and increased the sensitivity of the scFv ELISA microarray assays, although the anti-epitope precapture method introduced a risk of reagent transfer. Using the direct printing method, we show that scFv against prostate specific antigen (PSA) are highly specific when tested against 21 different IgG-based assays. In addition, the scFv microarray PSA assay gave comparable quantitative results to a commercial 96-well ELISA when tested using human serum samples. In addition, we find that thioredoxin-scFv fusions against other biomarkers of interest also have good lower limits of detection. Overall, these results suggest that minor modifications of the scFv construct are sufficient to produce reagents that are suitable for use in multiplex assay systems.