Strongyloidiosis by Strongyloides stercoralis is a disease of increasing interest in human and animal medicine. The scientific knowledge on canine strongyloidiosis is hindered by the poor diagnostics available. To assess the most sensitive and specific diagnostic method, feces and blood from 100 shelter dogs were screened for S. stercoralis by coprological, molecular and serological tests. Thirty-six dogs (36%) scored positive to S. stercoralis by coprology (22.3% to Baermann) and/or 30% to real time-polymerase chain reaction (rt-PCR). According to two composite reference standards (CRS) based on all coprological methods and rt-PCR (first CRS) or in combination with serology (second CRS), the most sensitive test was IFAT (93.8%; CI 82.8–98.7), followed by rt-PCR (80.6%; 95% CI 64–91.8) and Baermann (60.6%; 95% CI 42.1–77.1). The inconsistent shedding of L1 during the 4-week follow-up in infected dogs suggests the importance of multiple faecal collections for a reliable diagnosis. A combination of serological and coprological tests is recommended for the surveillance and diagnosis of S. stercoralis infection in dogs.