Prostate-specific antigen (PSA) is the most widely used biomarker for the early diagnosis of prostate cancer. Existing methods for PSA detection are burdened with some limitations and require improvement. Herein, we developed a novel microfluidic–electrochemical (μFEC) detection system for PSA detection. First, we constructed an electrochemical biosensor based on screen-printed electrodes (SPEs) with modification of gold nanoflowers (Au NFs) and DNA tetrahedron structural probes (TSPs), which showed great detection performance. Second, we fabricated microfluidic chips by DNA TSP-Au NF-modified SPEs and a PDMS layer with designed dense meandering microchannels. Finally, the μFEC detection system was achieved based on microfluidic chips integrated with the liquid automatic conveying unit and electrochemical detection platform. The μFEC system we developed acquired great detection performance for PSA detection in PBS solution. For PSA assays in spiked serum samples of the μFEC system, we obtained a linear dynamic range of 1–100 ng/mL with a limit of detection of 0.2 ng/mL and a total reaction time <25 min. Real serum samples of prostate cancer patients presented a strong correlation between the “gold-standard” chemiluminescence assays and the μFEC system. In terms of operation procedure, cost, and reaction time, our method was superior to the current methods for PSA detection and shows great potential for practical clinical application in the future.