Excretory/secretory (E/S) antigen derived from second-stage larvae of Toxocara canis maintained in defined medium in vitro has been well established worldwide for the immunodiagnosis of human toxocarosis by enzyme-linked immunosorbent assay. Such an enzyme-linked immunosorbent assay, based on the detection of human anti-T. canis (E/S antigen) serum immunoglobulin G, has recently been commercialized by Biokema-Affinity Products (Crissier-Lausanne, Switzerland). This commercial test kit was evaluated with regard to its application in a routine diagnostic laboratory and the reliability of the results. Of 78 patients with suspected clinical toxocarosis, 71 had anti-T. canis antibodies (positive serological result) corresponding to a diagnostic sensitivity of 91%; 14% of serum samples (n = 199) from patients with protozoan or with helminthic infections also showed positive reactions mainly related to infections with Trichinella, Strongyloides, and Fasciola species. An epidemiological study with 1,000 serum samples from randomly selected healthy blood donors and children in Switzerland demonstrated a seroprevalence of 2.7%. The test kit under evaluation had an overall diagnostic sensitivity of 91% and a relative specificity of 86%, the latter being related to some protozoan and helminthic infections. Because of the scarcity of such infections, potential cross-reactivity does not play a major role under the conditions found in the middle part of Europe. In conclusion, the application of the test kit provided for use in this study can be recommended for routine diagnostic use.