Porcine reproductive and respiratory syndrome virus (PRRSV) is an important swine pathogen, causing huge economic losses each year worldwide. Immunization with vaccines containing the glycoprotein 5 (GP5) of PRRSV is the main measure to induce neutralizing antibodies and control the disease. Here, we developed a GP5 protein-based ELISA for detecting antibodies against PRRSV. The overall yield of purified GP5 in E. coli flask culture was more than 45 mg/L cell culture. Western blot and IFA indicated that the GP5 protein was highly immunogenic. After optimization and validation with IDEXX PRRS using 566 clinical sera, the DSN, DSP, and accuracy of GP5-ELISA were 81.39%, 75.96%, and 80.39%, respectively. Besides, GP5-ELISA is highly specific, showing no cross-reactions with sera against other important swine pathogens. Hence, GP5 is a good diagnostic antigen and the GP5 protein-based ELISA has the potential to be used in the field.