We studied the use of the INNO-LIA syphilis score assay in the resolution of discordant positive screening results of the Murex ICE Syphilis enzyme immunoassay with the confirmatory results of both the Serodia Treponema pallidum particle agglutination and the fluorescent treponemal antibody-absorption assays, for the serological diagnosis of syphilis. This was an observational study on the serum samples received by the Syphilis Laboratory, Public Health Laboratory Services Branch, Centre for Health Protection, Department of Health, Hong Kong, during the period from January 2006 to December 2012. A total of 801 serum samples with discordant positive screening enzyme immunoassay results were used. Consensus results of such serum samples were derived from the most predominant results of the enzyme immunoassay, Treponema pallidum particle agglutination and fluorescent treponemal antibody-absorption assays. The age range of the individuals was 14 to 104 years (median of 52). There were 369 males and 432 females. The serum samples came from 305, 140 and 33 attendees of social hygiene clinics, maternal & child health centres and an integrated treatment centre, respectively, as well as 323 referral serum samples submitted by clinical microbiology laboratories of public hospitals in Hong Kong. There were 345 serum samples with positive consensus results, 100 with indeterminate consensus results and 356 with negative consensus results. Three serum samples with positive consensus results but invalid INNO-LIA syphilis score results and two with indeterminate consensus results but invalid INNO-LIA syphilis score results were excluded in the calculation of the proportions of specific agreement and the kappa coefficient. Of 378 serum samples, 139 showed agreement among positive results, 23 of 310 showed agreement among indeterminate results and 277 of 465 showed agreement among negative results. The proportions of agreement among positive, indeterminate and negative results were 0.37 (95% CI 0.32–0.42), 0.07 (95% CI 0.05–0.11) and 0.60 (95% CI 0.55–0.64), respectively. The kappa coefficient with quadratic weighting was 0.55 (95% CI 0.49–0.60). There were 60 serum samples with positive consensus results but negative INNO-LIA syphilis score results and 10 with negative consensus results but positive INNO-LIA syphilis score results. Although the INNO-LIA syphilis score assay can be considered a valid alternative confirmatory test for the serological diagnosis of syphilis, the present study showed that its use in the resolution of discordant positive screening enzyme immunoassay results was moderate. However, it was acknowledged that validity of the consensus results, which were derived from the most predominant results of the enzyme immunoassay, Treponema pallidum particle agglutination and fluorescent treponemal antibody-absorption assays, was uncertain. A more extensive characterization of the serum samples with discordant reactive screening treponemal test results is necessary.