Background: Strongyloides stercoralis is frequently underdiagnosed due to the limited sensitivity of stool-based methods. Serology improves detection, but venous blood collection and cold-chain requirements complicate field implementation. Dried blood spots (DBS) offer a practical alternative for large-scale surveys. This study aimed to evaluate the diagnostic accuracy of DBS-based serology and compare its performance with conventional ELISA. Methods: A cross-sectional study was conducted in peri-urban communities of Puerto Iguazú, Misiones, Argentina. Stool samples were examined using sedimentation–concentration and Baermann techniques. Venous blood on DBS was analyzed using a commercial ELISA (cELISA) and an in-house NIE-ELISA. Diagnostic accuracy was assessed using a primary reference standard. Agreement between serum and DBS was evaluated using kappa statistics, and ROC curves were generated for paired samples. Results: DBS cELISA showed the highest sensitivity (89.1%), followed by serum cELISA (81.1%) and in-house NIE-ELISA (71.7%). Concordance between sera vs DBS was substantial for cELISA (κ = 0.68) and fair for NIE-ELISA (κ = 0.21). ROC analysis showed comparable AUCs for cELISA using sera (0.85) and DBS (0.83), whereas in-house NIE-ELISA performance declined with DBS (AUC = 0.61). Conclusion: DBS-based cELISA demonstrated high diagnostic accuracy and strong agreement with sera, supporting its use as a field-adapted tool for strongyloidiasis surveillance. DBS serology represents a feasible and reliable alternative for epidemiological studies in resource-limited settings.