In an effort to develop biomarker-based diagnostics for preterm birth (PTB) risk, we created 3D printed microfluidic devices with multiplexed immunoaffinity monoliths to selectively extract multiple PTB biomarkers. The equilibrium dissociation constant for each monoclonal antibody toward its target PTB biomarker was determined. We confirmed the covalent attachment of three different individual antibodies to affinity monoliths using fluorescence imaging. Three different PTB biomarkers were successfully extracted from human blood serum using their respective single-antibody columns. Selective binding of each antibody toward its target biomarker was observed. Finally, we extracted and eluted three PTB biomarkers from depleted human blood serum in multiplexed immunoaffinity columns in 3D printed microfluidic devices. This is the first demonstration of multiplexed immunoaffinity extraction of PTB biomarkers in 3D printed microfluidic devices.