Background: This study compared the effects of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) and 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) as ⁶⁴Cu-chelating agents in newly developed prostate-specific membrane antigen (PSMA) target compounds, ⁶⁴Cu-cudotadipep and ⁶⁴Cu-cunotadipep, on pharmacokinetics. Methods: The in vitro stability of the chelators was evaluated using human and mouse serum. In vitro PSMA-binding affinity and cell uptake were compared using human 22Rv1 cells. To evaluate specific PSMA-expressing tumor-targeting efficiency, micro-positron emission tomography (mcroPET)/computed tomography (CT) and biodistribution analysis were performed using PSMA+ PC3-PIP and PSMA− PC3-flu tumor xenografts. Results: The serum stability of DOTA- or NOTA-conjugated ⁶⁴Cu-cudotadipep and ⁶⁴Cu-cunotadipep was >97%. The Ki value of the NOTA derivative, cunotadipep, in the in vitro affinity binding analysis was higher (2.17 ± 0.25 nM) than that of the DOTA derivative, cudotadipep (6.75 ± 0.42 nM). The cunotadipep exhibited a higher cellular uptake (6.02 ± 0.05%/1 × 10⁶ cells) compared with the cudotadipep (2.93 ± 0.06%/1 × 10⁶ cells). In the biodistribution analysis and microPET/CT imaging, the ⁶⁴Cu-labeled NOTA derivative, ⁶⁴Cu-cunotadipep, demonstrated a greater tumor uptake and lower liver uptake than the DOTA derivative. Conclusions: This study indicates that the PSMA-targeted ⁶⁴Cu-cunotadipep can be applied in clinical practice owing to its high diagnostic power for prostate cancer.