To the best of our knowledge, this was the first report on the integration of a signal amplification strategy into a microfluidic paper-based electrochemical immunodevice for the multiplexed measurement of cancer biomarkers. Signal amplification was achieved through the use of graphene to modify the immunodevice surface to accelerate the electron transfer and the use of silica nanoparticles as a tracing tag to label the signal antibodies. Accurate, rapid, simple, and inexpensive point-of-care electrochemical immunoassays were demonstrated using a photoresist-patterned microfluidic paper-based analytical device (μPAD). Using the horseradish peroxidase (HRP)-O-phenylenediamine-H2O2 electrochemical detection system, the potential clinical applicability of this immunodevice was demonstrated through its ability to identify four candidate cancer biomarkers in serum samples from cancer patients. The novel signal-amplified strategy proposed in this report greatly enhanced the sensitivity of the detection of cancer biomarkers. In addition, the electrochemical immunodevice exhibited good stability, reproducibility, and accuracy and thus had potential applications in clinical diagnostics.