Measurement of circulating protein biomarkers associated with disease can facilitate early detection, help guide treatment strategies and improve patient outcomes beyond current standards of care. The combination of inexpensive 3D-printed flow cells and electrochemical biosensors has recently emerged as a viable platform for low-cost, reliable biomarker measurements. Here, we report an electrochemical immunoassay system based on simple graphite electrode arrays, 3D-printed flow cells, and signal-generating magnetic bead bioconjugates for simultaneous detection of three biomarker proteins (cancer antigen 125 (CA-125), midkine (MK) and osteopontin (OPN)) associated with pediatric soft tissue sarcomas (PSTS). Magnetic bead bioconjugates are functionalized with large amounts of antibody and enzyme labels, electrode arrays are modified with gold nanoparticles and antibodies for specific capture of bioconjugate-labeled biomarkers, and 3D-printed flow cells facilitate their amperometric detection. Using this system, detection limits for CA-125, OPN and MK are 100 times lower than those obtained with commercial enzyme-linked immunosorbent assay (ELISA).