Purpose <p>Prostate-specific membrane antigen (PSMA) expression may decrease during the progression of prostate cancer (PCa), compromising the efficacy of PSMA-targeted imaging. Fibroblast activation protein (FAP), a key component of the tumor microenvironment, offers a complementary target. This study aimed to develop and evaluate PSMA/FAP bispecific tracers with a biphenyl scaffold for improved PET detection outcomes of PCa.</p> Methods <p>The binding affinities of precursors and their corresponding non-radioactive standards were assessed using recombinant PSMA and FAP proteins. PET/CT imaging was performed in 22Rv1 (PSMA-positive) and U87MG (FAP-positive) xenograft models. Clinical evaluation of [¹⁸F]AlF-NOTA-PSFA-1 was conducted in two PCa patients, directly compared with [⁶⁸Ga]Ga-PSMA-11.</p> Results <p>All precursors and their corresponding non-radioactive standards exhibited high affinity for both PSMA (<i>K</i>_i = 0.13–4.86 nM) and FAP (<i>K</i>_i = 0.012–0.40 nM). Among them, [¹⁸F]AlF-NOTA-PSFA-1, demonstrated the highest tumor uptake in both 22RV1 (SUV_max = 4.64) and U87MG (SUV_max = 7.23) models, along with favorable tumor retention over time. Compared to [⁶⁸Ga]Ga-PSMA-11, [¹⁸F]AlF-NOTA-PSFA-1 showed higher lesion uptake, reduced background accumulation in off-target organs, and improved imaging quality in metastatic PCa patient.</p> Conclusions <p>The biphenyl-based bispecific tracers developed in this study achieved high tumor affinity, prolonged retention, and notably reduced renal uptake in mouse models. [¹⁸F]AlF-NOTA-PSFA-1 emerged as the most promising candidate, demonstrating superior imaging performance over [⁶⁸Ga]Ga-PSMA-11 in both preclinical and first-in-human evaluation.</p> Trial registration <p>Clinical trial registry NCT06690970, Registered 14 Nov 2024.</p>