Chlamydia trachomatis (Ct) serology is an important tool to support infection and disease surveillance of the population but there are currently no formal reference reagents and few commercial assays available. Our objective was to develop an internally standardized ELISA capable of estimating antibody levels against the virulence factor antigen, pgp3, that could be used for Ct serosurveillance. Purified trimeric pgp3 antigen was stable and the internal standard, quality controls, intra- and inter-assay repeatability metrics were robust. For example, retesting a panel of serum samples (n = 203) resulted in 12 % and 13 % seropositivity for the initial and repeat tests, respectively (Kappa 0.933, 95 %CI 0.857-1.000). A comparison of serum samples from children who would not be expected to have had exposure to Ct and samples from females with a history of chlamydia infection, though not necessarily antibody positive at the time of sampling, generated excellent specificity (99.4 %) and adequate sensitivity (67.9 %) for surveillance purposes. The pgp3 ELISA reports results in antibody levels (AU/mL) which are amenable to recalibration should an international reference reagent be approved in the future.