The aim of this study was to develop a blocking enzyme-linked immunosorbent assay (bELISA) based on a biotinylated nanobody target the S1 protein of porcine epidemic diarrhea virus (PEDV) for detecting the anti-PEDV antibodies and evaluating the immune effect of the vaccine. The gene encoding the single-domain antibody sdAb3 target the PEDV S1 protein was amplified and the Avitag sequence was fused at its 3′-end. The PCR product was cloned into the expression vector pET-21b for expression and purification of the sdAb3-Avitag protein. The purified sdAb3-Avitag fusion protein was biotinylated and its activity was determined. Using the recombinant S1 protein as a coating antigen, a bELISA was established and optimized. Serum samples were tested in parallel by the bELISA and a commercial kit. The recombinant vector pET21b-sdAb3-Avitag was constructed to express the tagged sdAb3. After induction for expression, the biotin-labeled sdAb3 (sdAb3-Biotin) with high purity and good activity was obtained. For the optimized bELISA, the coating concentration of the S1 protein was 200 ng/well, the serum dilution was 1:2 and incubated for 2 h, the dilution ratio of the biotinylated sdAb3 was 1:8 000 and incubated for 30 min, the dilution of the enzyme-labeled antibody was 1:5 000 and incubated for 30 min. The bELISA had no cross reaction with the sera of major porcine viruses including transmissible gastroenteritis virus, porcine reproductive and respiratory syndrome virus and showed good specificity and reproducibility. For a total of 54 porcine serum samples tested, the overall compliance rate of the bELISA with a commercial kit was 92.56%. This study developed a rapid and reliable bELISA method, which can be used for serosurveillance and vaccine evaluation for PEDV.