(1) Background: Autoantibodies to tumor-associated antigens (TAAs) have emerged as promising cancer biomarkers. Luminex technology offers a powerful approach for the simultaneous detection of multiple anti-TAA autoantibodies. (2) Methods: We aimed to utilize Luminex technology to evaluate and optimize a panel of anti-TAAs autoantibodies for detecting prostate cancer (PCa), which included autoantibodies to fourteen TAAs. A total of 163 serum samples (91 PCa, 72 normal controls) were screened to determine the levels of the autoantibodies using the Luminex assay. (3) Results: Twelve autoantibodies exhibited significantly high frequencies ranging from 19.8% to 51.6% in the PCa group. Receiver operating characteristic (ROC) curve analysis revealed area under the curve (AUC) values ranging from 0.609 to 0.868 for the twelve autoantibodies individually. We further confirmed the performance of the HSP60 autoantibody by using an enzyme-linked immunosorbent assay (ELISA) in a larger sample comprising 200 PCa sera, 20 benign prostatic hyperplasia (BPH) sera, and 137 normal control sera. The results obtained from the Luminex assay were consistent with the ELISA findings. We developed a panel consisting of three autoantibodies (p16, IMP2, and HSP60) which achieved an impressive AUC of 0.910 with a sensitivity of 71.4% and a specificity of 95.8%. The panel was also evaluated in PCa patients from different races/ethnicities with the best performance observed in distinguishing the Hispanic American patients with PCa from normal controls. (4) Conclusions: We developed an anti-TAA autoantibody panel for the detection of PCa that exhibits promising performance. This panel holds significant potential as a high-throughput tool to facilitate PCa detection.