Purpose <p>Activation of androgen receptor (AR) signaling is a hallmark of prostate cancer. Dynamic changes in AR expression exacerbate AR heterogeneity throughout prostate cancer therapy. This study aims to develop a series of ⁶⁸Ga-labeled Enzalutamide-based positron emission tomography (PET) tracers for AR imaging.</p> Methods <p>[⁶⁸Ga]Ga-DOTA-FZAR-1, [⁶⁸Ga]Ga-DOTA-FZAR-2, and [⁶⁸Ga]Ga-DOTAGA-FZAR-3 were synthesized and the stability was analyzed in vitro. The AR specificity of the three radiotracers was assessed in vitro using AR-negative and AR-positive prostate cancer cell lines and in vivo using tumor xenograft-bearing mice. Moreover, the first-in-human evaluation of [⁶⁸Ga]Ga-DOTA-FZAR-2 was conducted in eight patients with prostate cancer.</p> Results <p>[⁶⁸Ga]Ga-DOTA-FZAR-1, [⁶⁸Ga]Ga-DOTA-FZAR-2, and [⁶⁸Ga]Ga-DOTAGA-FZAR-3 were successfully synthesized with a radiochemical purity of more than 99%, and had good stability in vitro. Cellular uptake assays revealed that the radiotracers had the highest, intermediate, and lowest uptake in LNCaP, 22Rv1, and PC-3 cells, respectively, strongly correlating with AR expression levels (<i>P</i> &lt; 0.001). Consistent with cellular uptake, the radiotracers also exhibited a hierarchical uptake pattern (highest to lowest) in tumors of mice bearing LNCaP, 22Rv1 and PC-3 xenografts, respectively. In addition, all three radiotracers were primarily eliminated through the urinary system, as confirmed by ex vivo biodistribution studies. More importantly, first-in-human investigation showed safety and diagnostic value of [⁶⁸Ga]Ga-DOTA-FZAR-2 in AR-associated prostate cancer patients.</p> Conclusion <p>We developed and validated a series of ⁶⁸Ga-labeled Enzalutamide-based PET tracers for AR imaging. Initial preclinical and clinical evidence indicate that [⁶⁸Ga]Ga-DOTA-FZAR-2 enables noninvasive, whole-body, and dynamic monitoring of AR expression in prostate cancer patients throughout therapy.</p>