Prostate specific antigen (PSA) is a widely used marker for the diagnosis of prostate cancer, and the increasing attention has been attracted on the development of rapid assay using biosensing technology. However, it remains challenging for the sensitive and selective detection of PSA in clinical samples. Here, we report a label-free microfluidic paper-based analytical device for highly sensitive electrochemical detection of PSA. The paper device was fabricated with wax printing to generate hydrophobic and hydrophilic layers for the construction of microfluidic channel, followed by screen-printing of three electrodes including working, counter and reference electrode. Gold nanoparticles (AuNPs)/reduced graphene oxide (rGO)/thionine (THI) nano composites were synthesized and characterized, which were coated onto working electrodes for the immobilization of DNA aptamer probe. THI servers as the electrochemical mediator to transduce the biological recognition between DNA aptamer and PSA, and the excellent conductivity of AuNPs and rGO also play a significant role of electron transfer, leading to a sensitive detection for PSA, able to detect PSA as low as 10 pg mL-1, with a linear range from 0.05 to 200 ng mL-1. We demonstrated that our electrochemical sensor for the detection of clinical serum samples, indicating that our sensor would provide a new platform for low cost, sensitive and point-of-care diagnosis of prostate cancer.