Although cardiovascular diseases such as heart failure (HF) affect 30 million people globally, the early detection of HF has, until recently, been difficult and prone to misdiagnoses. Monitoring the circulatory levels of a relatively new biomarker, the N-terminal prohormone of a B-type natriuretic peptide, could be used for early risk evaluation of HF. Therefore, we developed a pneumatically-driven, automatic integrated microfluidic platform equipped with micromixers, micropumps, and microvalves for the simultaneous detection of NT-proBNP in up to six clinical samples within 25 min by using a novel aptamer-based sandwich assay, and the limit of detection was only 1.53 pg mL-1; given that the chip is 64% more compact than those developed in our prior works and requires only 5 μL of sample input, it may serve as a promising tool for early diagnosis of HF.