The burden of disease and associated healthcare costs of syphilis are significant despite widespread screening and treatment. Our objective was to conduct an economic evaluation using a simulation model when comparing enzyme immunoassay (EIA) initial testing and Inno-Lia (IL) confirmatory testing (EIA + IL) with rapid plasma reagin (RPR) initial testing and Treponema pallidum particle agglutination assay (TPPA) and fluorescent treponemal antibody absorption assay (FTA-ABS) confirmatory testing (RPR + TPPA/FTA). Estimates of prevalence, test costs and utilization of services for 2006 were derived from Alberta databases. Estimates of test characteristics were derived from the available literature. The incremental cost-effectiveness ratio was Canadian $461 per additional correct diagnosis (less costly and more effective). EIA + IL is cost-effective when compared with RPR + TPPA/FTA for screening and diagnosis of syphilis.