In this work, we developed giant magnetoresistive (GMR) based handheld biosensing systems that serve as platform for detecting human NT-proBNP. This assay takes advantages of high sensitivity and real-time signal readout of GMR biosensor. The limit of detection was estimated to be less than 0.01ng/mL, and detection range covered from 0.01 ng/mL to 5 ng/mL was obtained. The assay can be completed within 20 min, which is very important for further development of point-of-care testing. The proposed GMR handheld system is also successfully used for the detection of real NT-proBNP human samples. It can be foreseen that this handheld detection system could become a robust contender in the applications of in vitro biomarker diagnostics.