TGF-beta3 has been implicated in the pathology of ocular diseases, but its concentration in human aqueous humor has never been assessed. In this study, we established an enzyme-linked immunosorbent assay (ELISA) for TGF-beta3 and quantitated it in aqueous humor collected from patients with pseudoexfoliation syndrome (PE), primary open angle glaucoma, chronic angle closure glaucoma and cataract (as the control). To develop the TGF-beta3 ELISA, we screened antibodies to identify the best combination, validated the assay for aqueous humor, and optimized the procedure for preparing activated TGF-beta3. As a result, our ELISA was highly selective and reproducible. Using our ELISA, we discovered a significantly elevated concentration of TGF-beta3 in PE eyes. We also developed new TGF-beta1 and -beta2 ELISAs, and were able, for the first time, to quantitate all the TGF-beta isoforms in the aqueous humor from a single eye, to assess their proportional effects on the pathogenesis of ocular diseases.