Sensitive and selective detection for cancer biomarkers are critical in cancer clinical diagnostics. Here we developed a microfluidic protein chip for an ultrasensitive and multiplexed assay of cancer biomarkers. Aqueous-phase-synthesized CdTe/CdS quantum dots (aqQDs) were employed as fluorescent signal amplifiers to improve the detection sensitivity. Secondary antibodies (goat anti-mouse IgG) were conjugated to luminescent CdTe/CdS QDs to realize a versatile fluorescent probe that could be used for multiplexed detection in both sandwich and reverse phase immunoassays. We found that our microfluidic protein chip not only possessed ultrahigh femtomolar sensitivity for cancer biomarkers, but was selective enough to be directly used in serum. This protein chip thus combines the high-throughput capabilities of a microfluidic network with the high sensitivity and multicolor imaging ability offered by highly fluorescent QDs, which can become a promising diagnostic tool in clinical applications.