In this work, we report a simple and novel electrochemical multiplexed immunosensor on a flexible polydimethylsiloxane (PDMS) slice deposited with 8 × 8 nano-Au film electrodes for simultaneous detection of prostate specific antigen (PSA), prostate specific membrane antigen (PSMA), and interleukin-6 (IL-6). Primary antibodies linked with magnetic beads (Ab1-MBs) were modified on the nano-Au film electrodes via magnetic force. In the presence of corresponding antigen, horse radish peroxidase-secondary antibody-conjugated gold nanorods (HRP-Ab2-gold NRs) were brought into the surface of electrodes, generating obvious electrochemical signals of H2O2 reduction reactions. Based on this, the designed immunosensor provide good performance in sensitivity and specificity during the detection of above three biomarkers for prostate cancer. The electrochemical multiplexed immunosensor was verified for selective and accurate detection of complex samples in human serum. Data suggested that the reported multiplexed immunosensing strategy holds great promise for applications in clinical assay and diseases diagnosis.