Although PSMA PET/CT imaging has great potential for noninvasively detecting prostate cancer (PCa), limitations exist for patients with low PSMA expression, caused by androgen deprivation treatment or neuroendocrine differentiation. Analysis of The Cancer Genome Atlas Prostate Adenocarcinoma (TCGA-PRAD) data found that erythropoietin-producing hepatocellular receptor A2 (EphA2), a receptor overexpressed in most PCa could be a potential target for PSMA-negative PCa. A fluorescent ligand ETF and a radiolabeled ligand [18F]AlF-ETN derived from a EphA2-targeting bicyclic peptide were synthesized and investigated. ETF could selectively stain and visualize the EphA2-positive but PSMA-negative PC3 cells, in complementary to the PSMA-targeting probe. PET/CT imaging and biodistribution experiments demonstrated that [18F]AlF-ETN specifically accumulated in PC3 tumors with a high contrast (tumor-to-muscle ratio: 21.29 ± 6.55). In conclusion, we have demonstrated the potential for using EphA2 to detect PSMA-negative PCa and developed a radiolabeled ligand [18F]AlF-ETN to specifically image EphA2 expressing PCa with high contrast.